bluuebunny/crossref_metadata_2025 huggingface dataset:
Esophagogastric junction distensibility: a factor contributing to sphincter incompetence
Authors: Pandolfino John E. , Shi Guoxiang , Curry Jennifer , Joehl Raymond J. , Brasseur James G. , Kahrilas Peter J.
Published: March 2015

To quantify the effect of hiatus hernia (HH) on esophagogastric junction (EGJ) distensibility, eight normal subjects and nine gastroesophageal reflux disease (GERD) patients with HH were studied with concurrent manometry, fluoroscopy, and stepwise controlled barostatic distention of the EGJ. The minimal barostatic pressure required to open the EGJ during the interswallow period was determined. Thereafter, barium swallows were imaged in 5-mmHg increments of intrabag pressure. EGJ diameter and length were measured at each pressure during deglutitive relaxation. The EGJ opening diameter was greater in hernia patients compared with normal subjects during deglutitive relaxation at all pressures, and EGJ length was 23% shorter. EGJ opening pressure among hernia patients was lower than normal subjects during the interswallow period. In conclusion, the EGJ of GERD patients with HH was more distensible and shorter than normal subjects. These findings partially explain why HH patients are predisposed to reflux by mechanisms other than transient lower esophageal sphincter relaxations, sustain greater volumes of refluxate, and have a reduced ability to discriminate gas from liquid reflux.
Hypergastrinemia in response to gastric inflammation suppresses somatostatin
Authors: Zavros Yana , Rieder Gabriele , Ferguson Amy , Samuelson Linda C. , Merchant Juanita L.
Published: March 2015

Hypergastrinemia and a reduction in tissue somatostatin occur in Helicobacter pylori-infected patients. We investigated whether the D cell may be a direct target of gastric inflammation and hypergastrinemia. D cells were quantified by morphometry and flow cytometry in 16-wk-old wild-type (G+/+) and gastrin-deficient (G−/−) mice. Hypochlorhydric G−/− mice were treated with either antibiotics for 20 days or infused with gastrin (G-17) for 14 days. G+/+ mice were made hypochlorhydric by treating them with omeprazole for 2 mo. G−/− mice showed significant inflammation compared with the G+/+ mice, which resolved after 20 days of antibiotic treatment. D cell numbers were not significantly different between G−/− and G+/+ mice. After G-17 was infused, fundic and antral D cell numbers decreased in the G−/− mice. G+/+ animals made hypergastrinemic with omeprazole exhibited decreased D cell numbers. When omeprazole-treated mice were treated with antibiotics alone, elevated plasma gastrin levels returned to baseline and D cell numbers returned to resting levels despite persistent hypochlorhydria. Hypergastrinemia, induced by inflammation, results in decreased D cell numbers. Thus the stomach responds to the presence of inflammation by reducing somatostatin levels, thereby releasing the inhibition on the G and parietal cells to maximize gastric acid output.
Age-associated loss of heterozygosity of tumor suppressor genes in the gastric mucosa of humans
Authors: Moragoda Lathika , Jaszewski Richard , Kulkarni Prasad , Majumdar Adhip P. N.
Published: March 2015

The current study is based on the hypothesis that aging predisposes gastric mucosa to carcinogenesis through altered expression and/or mutations of genes involved in cell growth. To test this hypothesis, we investigated the age-associated changes in mutation of adenomatous polyposis coli ( APC), deleted in colorectal cancer ( DCC), p53, and K- ras genes in the gastric mucosa of 19 healthy subjects of varying ages (25–91 yr). Specifically, we studied the loss of heterozygosity (LOH) of these genes in cardia, body, and antrum of the stomach. We observed that 3 of 19 subjects (16%) over 60 yr of age show LOH of at least one of the tumor suppressor genes. Among the subjects over 60 yr of age, the incidence of LOH is 38% (3/8). Two of three subjects had mutations in more than one tumor suppressor gene. In all three affected subjects, mutation in APC, DCC, or p53 was located mainly in the body of the stomach, suggesting increased susceptibility of this region to neoplastic changes. However, no LOH of K- ras was observed in these subjects. Our observation that subjects over 60 yr of age show mutation in one or more of the tumor suppressor genes suggests an age-related increase in predisposition of the stomach to neoplasia.
Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine
Authors: Rost Daniel , Mahner Sven , Sugiyama Yuichi , Stremmel Wolfgang
Published: March 2015

Multidrug resistance-associated protein 3 (MRP3; symbol ABCC3), has been shown to mediate ATP-dependent transport of organic anions including 17β-glucuronosyl estradiol, glucuronosyl bilirubin, monovalent, and sulfated bile salts. MRP3 mRNA expression was reported in rat intestine suggesting a role of MRP3 in the intestinal transport. We examined the expression and localization of MRP3 in rat small and large intestine by RT-PCR, immunofluorescence, and immunoblot analysis. MRP3 was identified in all intestinal segments by RT-PCR. MRP3 expression was low in duodenum and jejunum but markedly increased in ileum and colon. With the use of a rat MRP3 specific antibody, MRP3 was localized to the basolateral domains of enterocytes. Immunofluorescence analysis and immunoblot analysis confirmed a strong expression of rat MRP3 in ileum and colon. In contrast, MRP2 was predominantly expressed in the proximal segments of rat small intestine. Our findings demonstrate a high expression of rat MRP3 in ileum and colon and provide evidence for an involvement of MRP3 in the ATP-dependent transport of organic anions, including bile salts from the enterocyte into blood.
Ca2+sparks and BK currents in gallbladder myocytes: role in CCK-induced response
Authors: Pozo María J. , Pérez Guillermo J. , Nelson Mark T. , Mawe Gary M.
Published: March 2015

We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized Ca2+release events (sparks) and large-conductance Ca2+-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 μM). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 ± 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway.
Novel MUC1 splice variants contribute to mucin overexpression in CFTR-deficient mice
Authors: Hinojosa-Kurtzberg A. Marina , Johansson Malin E. V. , Madsen Cathy S. , Hansson Gunnar C. , Gendler Sandra J.
Published: March 2015

A cystic fibrosis (CF) mouse expressing the human mucin MUC1 transgene (CFM) reverted the CF/Muc1−/−phenotype (little mucus accumulated in the intestine) to that of CF mice expressing mouse Muc1, which exhibited increased mucus accumulation. Western blots and immunohistochemical analysis showed that the MUC1 protein was markedly increased in CFM mice in which it was both membrane bound and secreted into the intestinal lumen. Studies to determine the reason for increased levels of the extracellular domain of MUC1 mucin identified mRNA and protein of two novel splice variants and the previously described secreted MUC1 lacking the cytoplasmic tail (MUC1/SEC). Novel MUC1 splice variants, CT80 and CT58, were both transmembrane proteins with cytoplasmic tails different from the normal MUC1. The MUC1-CT80 and MUC1/SEC forms are found expressed mainly in the CFM mice intestines. Thus MUC1 expression is increased, and it appears that alternate cytoplasmic tails may change its role in signaling. MUC1 could be an important contributor to the CF intestinal phenotype.
ERK1/2 and Egr-1 contribute to increased TNF-α production in rat Kupffer cells after chronic ethanol feeding
Authors: Kishore Raj , Hill Jeanette R. , McMullen Megan R. , Frenkel Julia , Nagy Laura E.
Published: March 2015

Activation of Kupffer cells by lipopolysaccharide (LPS) is a critical step in the pathogenesis of alcoholic liver disease. Kupffer cells isolated from rats fed ethanol in their diet for 4 wk accumulated 4.3-fold more tumor necrosis factor (TNF)-α in response to LPS compared with pair-fed rats. In contrast, LPS-stimulated interleukin (IL)-1 accumulation was 50% lower after ethanol feeding. LPS-stimulated TNF-α mRNA accumulation was twofold higher after ethanol feeding, whereas IL-1β mRNA accumulation was blunted. To understand the mechanisms for this differential response, we investigated the effects of ethanol on LPS-dependent signal transduction. Chronic ethanol feeding increased LPS-stimulated extracellular receptor-activated kinases 1/2 (ERK1/2) activation. Activation of ERK1/2 was required for maximal increases in TNF-α and IL-1β mRNA and was associated with increased binding of early growth response-1 (Egr-1) to the TNF-α promoter after ethanol feeding. In contrast, ethanol feeding completely abrogated activation of nuclear factor-κB DNA-binding activity by LPS and had no effect on AP-1 binding. Together, these data suggest that enhanced activation of ERK1/2 and Egr-1 contributes to increased TNF-α production after chronic ethanol feeding.
Active K+secretion through multiple KCa-type channels and regulation by IKCachannels in rat proximal colon
Authors: Joiner William J. , Basavappa Srisaila , Vidyasagar Sadasivan , Nehrke Keith , Krishnan Selvi , Binder Henry J. , Boulpaep Emile L. , Rajendran Vazhaikkurichi M.
Published: March 2015

Colonic K+secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca2+. However, the precise mechanisms by which this rise in Ca2+leads to K+efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] Ca2+-activated K+channels. In dietary K+-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K+channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca2+-activated K+channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K+secretion was studied on epithelial strips of rat proximal colon using unidirectional86Rb+fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net86Rb+secretion. The mucosal addition of CLT completely inhibited carbachol-induced net86Rb+secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K+, carbachol-induced86Rb+secretion was abolished in dietary K+-depleted animals. These results suggest that the rSK4 channel mediates K+secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent K+loss during dietary K+depletion.
Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line
Authors: Cho Han J. , Kim Woo K. , Kim Eun J. , Jung Kyeong C. , Park Soochul , Lee Hyun S. , Tyner Angela L. , Park Jung H. Y.
Published: March 2015

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-α led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-α-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.
Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis
Authors: Halpern Melissa D. , Holubec Hana , Dominguez Jessica A. , Meza Yolanda G. , Williams Catherine S. , Ruth Miriam C. , McCuskey Robert S. , Dvorak Bohuslav
Published: March 2015

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by mutisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-α, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-α levels were also measured. Production of hepatic IL-18 and TNF-α and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-α in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.
PGF2α-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle
Authors: Cao Weibiao , Harnett Karen M. , Behar Jose , Biancani Piero
Published: March 2015

Lower esophageal sphincter (LES) tone depends on PGF2αand thromboxane A2acting on receptors linked to Gi3and Gqto activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF2αsignal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of G13and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [35S]GTPγS-binding assay confirmed that G13, RhoA, and ARF1 were activated by PGF2α. Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of Gqand Gi3, and a [35S]GTPγS-binding assay confirmed that Gqand Gi3were activated by PGF2α. PGF2α-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF2αconcentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF2αactivates a PLD- and protein kinase C (PKC)-dependent pathway through G13, RhoA, and ARF1. In LES, PGF2αreceptors are coupled to Gqand Gi3, activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF2αactivates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.
Reciprocal activity of longitudinal and circular muscle during intestinal peristaltic reflex
Authors: Grider J. R.
Published: March 2015

A two-compartment, flat-sheet preparation of rat colon was devised, which enabled exclusive measurement of longitudinal muscle activity during the ascending and descending phases of the peristaltic reflex. A previous study using longitudinal muscle strips revealed the operation of an integrated neuronal circuit consisting of somatostatin, opioid, and VIP/pituitary adenylate cyclase-activating peptide (PACAP)/nitric oxide synthase (NOS) interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle strips that could lead to descending contraction and ascending relaxation of this muscle layer. Previous studies in peristaltic preparations have also shown that an increase in somatostatin release during the descending phase causes a decrease in Met-enkephalin release and suppression of the inhibitory effect of Met-enkephalin on VIP/PACAP/NOS motor neurons innervating circular muscle and a distinct set of VIP/PACAP/NOS interneurons. The present study showed that in contrast to circular muscle, longitudinal muscle contracted during the descending phase and relaxed during the ascending phase. Somatostatin antiserum inhibited descending contraction and augmented ascending relaxation of longitudinal muscle, whereas naloxone had the opposite effect. VIP and PACAP antagonists inhibited descending contraction of longitudinal muscle and augmented ascending relaxation. Atropine and tachykinin antagonists inhibited descending contraction of longitudinal muscle. As shown in earlier studies, the same antagonists and antisera produced opposite effects on circular muscle. We conclude that longitudinal muscle contracts and relaxes in reverse fashion to circular muscle during the peristaltic reflex. Longitudinal muscle activity is regulated by excitatory VIP/PACAP/NOS interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle.
Relative contribution of various airway protective mechanisms to prevention of aspiration during swallowing
Authors: Medda Bidyut K. , Kern Mark , Ren Junlong , Xie Pengyan , Ulualp Seckin O. , Lang Ivan M. , Shaker Reza
Published: March 2015

Deglutitive airway protective mechanisms include glottal closure, epiglottal descent, and anterosuperior displacement of the larynx. Aspiration of swallowed material may occur during the pre-, intra-, or postpharyngeal phase of swallowing. Our objectives were to determine the relative contribution of the airway protective mechanisms during each phase of swallow in 14 decerebrated cats before and after suprahyoid myotomy, epiglottectomy, and unilateral cordectomy. After myotomy, superior excursions of the hyoid, thyroid, and cricoid cartilages and anteroposterior diameter of maximum upper esophageal spincter (UES) opening were significantly diminished, but the incidence of pharyngeal residue significantly increased ( P < 0.05). No aspiration was observed in the predeglutitive period. After myotomy, the incidence of aspiration significantly increased in both intra- and postdeglutitive periods. Epiglottectomy did not alter aspiration incidence, but unilateral cordectomy resulted in a 100% incidence of intra- and postdeglutitive aspiration. In conclusion, glottal closure constitutes the primary mechanism for prevention of intra- and postdeglutitive aspiration, but laryngeal elevation may assist this function. Bolus pulsion without laryngeal distraction can open the UES, but at risk of aspiration due to decreased pharyngeal clearance. The epiglottis provides no apparent airway protection during any phase of swallowing.
Modulation of intestinal protein synthesis and protease mRNA by luminal and systemic nutrients
Authors: Adegoke Olasunkanmi A. J. , McBurney Michael I. , Samuels Susan E. , Baracos Vickie E.
Published: March 2015

Route of nutrient supply is important in regulation of intestinal protein metabolism, because total parenteral nutrition, compared with enteral feeding, leads to profound atrophy. Participation of the fractional rate of protein synthesis ( Ks), their degradation in regulation of gut protein balance, and their possible modulation by specific nutrients are the focus of our work. We developed an in situ experimental system that allows controlled exposure of intestinal mucosa to nutrients systemically, luminally, or both. We examined the effects of systemic glucose and amino acid (AA) infusion in overnight-fasted piglets. Jejunal segments within each piglet were simultaneously, luminally perfused with solutions containing various AAs or glucose. Intravenous infusion of glucose increased mucosal Ks by 16% ( P < 0.05), whereas intravenous infusion of AA had no effect on Ks. Systemic glucose infusion had no effect on mRNA levels for components of the ubiquitin-proteasome proteolytic pathway. However, levels of these mRNA were reduced by intravenous or luminal AA supply. This effect was greatest (−50%) when highest tissue concentrations of AAs were achieved by the simultaneous infusion of AA by both routes ( P < 0.05). Our findings suggest that not only is the modulation of protein balance in the intestine in response to nutrients in part attributable to anabolic stimulation of protein synthesis initiated by the systemic appearance of glucose, but a fall in protein degradation is also a likely contributor. AAs appear to be a key factor required to reduce expression of genes connected with proteolysis.
ANG II stimulates PKC-dependent ERK activation, DNA synthesis, and cell division in intestinal epithelial cells
Authors: Chiu Terence , Santiskulvong Chintda , Rozengurt Enrique
Published: March 2015

PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation and carcinogenesis. With the use of IEC-18 cells arrested in G0/G1, addition of phorbol esters resulted in a modest increase in [3H]thymidine incorporation and a slight shift toward the S and G2/M phases of the cell cycle, whereas the combination of EGF and phorbol 12,13-dibutyrate (PDB) synergistically stimulated DNA synthesis. To investigate the effects of receptor-mediated PKC activation on mitogenesis, we demonstrated that ANG II induced ERK activation, a response completely blocked by pretreatment with mitogen/extracellular signal-regulated kinase inhibitors or specific PKC inhibitors. Furthermore, ANG II stimulated an over threefold increase in [3H]thymidine incorporation that was corroborated by flow cytometric analysis of the cell cycle to levels comparable to that achieved by the combination of EGF and PDB. Taken together, our results indicate that receptor-mediated PKC activation, as induced by ANG II, transduces mitogenic signals leading to DNA synthesis and cell proliferation in IEC-18 cells.
Peripheral CRF activates myenteric neurons in the proximal colon through CRF1 receptor in conscious rats
Authors: Miampamba Marcel , Maillot Celine , Million Mulugeta , Taché Yvette
Published: March 2015

Corticotropin-releasing factor (CRF) injected peripherally induces clustered spike-burst activity in the proximal colon through CRF1 receptors in rats. We investigated the effect of intraperitoneal CRF on proximal colon ganglionic myenteric cell activity in conscious rats using Fos immunohistochemistry on the colonic longitudinal muscle/myenteric plexus whole mount preparation. In vehicle-pretreated rats, there were only a few Fos immunoreactive (IR) cells per ganglion (1.2 ± 0.6). CRF (10 μg/kg ip) induced Fos expression in 19.6 ± 2.1 cells/ganglion. The CRF1/CRF2 antagonist astressin (33 μg/kg ip) and the selective CRF1 antagonist CP-154,526 (20 mg/kg sc) prevented intraperitoneal CRF-induced Fos expression in the proximal colon (number of Fos-IR cells/ganglion: 2.7 ± 1.2 and 1.0 ± 1.0, respectively), whereas atropine (1 mg/kg sc) had no effect. Double labeling of Fos with protein gene product 9.5 revealed the neuronal identity of activated cells that were encircled by varicose fibers immunoreactive to vesicular acetylcholine transporter. Fos immunoreactivity was mainly present in choline acetyltransferase-IR nerve cell bodies but not in the NADPH-diaphorase-positive cells. These results indicate that peripheral CRF activates myenteric cholinergic neurons in the proximal colon through CRF1 receptor.
Modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly by butyrate
Authors: Marcil Valérie , Delvin Edgard , Seidman Ernest , Poitras Lucie , Zoltowska Monika , Garofalo Carole , Levy Emile
Published: March 2015

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P< 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly ( P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.
Neuronal locus and cellular signaling for stimulation of ileal giant migrating and phasic contractions
Authors: Sarna Sushil K.
Published: March 2015

We investigated the neuronal locus, the role of PKC activation, and utilization of extracellular Ca2+ and intracellular Ca2+ release in smooth muscle cells for the generation of giant migrating contractions (GMCs) and rhythmic phasic contractions (RPCs) in intact normal and inflamed canine ileum. Calcitonin gene-related peptide (CGRP), administered close intra-arterially, stimulated GMCs at higher doses and RPCs at smaller doses. These effects were blocked by prior close intra-arterial infusions of CGRP8–37, atropine, hexamethonium, and TTX but not by tachykinin, serotonin, and histaminergic receptor subtype antagonists. Both types of contractions were blocked by verapamil in normal and inflamed ileums. Dantrolene and ruthenium red blocked only the RPCs in normal ileum but blocked both GMCs and RPCs in the inflamed ileum. PKC inhibition by chelerythrine blocked GMCs only in inflamed ileum but blocked RPCs in both normal and inflamed ileums. The inhibition of phospholipase C by neomycin blocked both RPCs and GMCs in normal and inflamed ileums. In conclusion, acetylcholine is the common neurotransmitter for the stimulation of both GMCs and RPCs, but the signaling cascades for their stimulation are partially divergent, and they differ also in the normal and inflamed states.
Identification of a basolateral Cl−/HCO 3 − exchanger specific to gastric parietal cells
Authors: Petrovic Snezana , Ju Xie , Barone Sharon , Seidler Ursula , Alper Seth L. , Lohi Hannes , Kere Juha , Soleimani Manoocher
Published: March 2015

The basolateral Cl−/HCO[Formula: see text] exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H+-K+-ATPase. Here, we report the identification of a new Cl−/HCO[Formula: see text]exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl−/HCO[Formula: see text] exchanger that is active in both acidic and alkaline pHi. On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl−/HCO[Formula: see text] exchanger in gastric parietal cells and plays a major role in gastric acid secretion.
Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA
Authors: Murthy Karnam S. , Zhou Huiping , Grider John R. , Makhlouf Gabriel M.
Published: March 2015

The role of RhoA in myosin light-chain (MLC)20dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active RhoV14, and phosphorylation site-deficient RhoA188. Activators of PKA (5,6-dichloro-1-β-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP) induced phosphorylation of constitutively active RhoV14and agonist (ACh)- or GTPγS-stimulated wild-type RhoA but not RhoA188. Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and RhoV14to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/Rho kinase association, MLC20phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration (1 μM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant RhoA188, MLC20phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC20and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.
Mechanism of staurosporine-induced apoptosis in murine hepatocytes
Authors: Feng Guoping , Kaplowitz Neil
Published: March 2015

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-α-induced apoptosis. STS activated IκB kinase and nuclear factor-κB (NF-κB) nuclear translocation and DNA binding but inhibited transactivation of IκB-α, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-κB reporter in transfected Huh-7 cells.
Intestinal alkaline phosphatase release is not associated with chylomicron formation
Authors: Nauli Andromeda M. , Zheng Shuqin , Yang Qing , Li Ronggui , Jandacek Ronald , Tso Patrick
Published: March 2015

Intestinal alkaline phosphatase (IAP) is one of the major sources of alkaline phosphatase in circulation. It is secreted into the intestinal lumen, serum, and lymph. After the ingestion of lipid, lymphatic alkaline phosphatase secretion increases significantly. We have found that the nonabsorbable fat olestra is unable to stimulate lymphatic alkaline phosphatase secretion. We also found that the hydrophobic surfactant Pluronic L-81, which blocks chylomicron formation, fails to inhibit this increase in lymphatic alkaline phosphatase secretion. These results suggest that it is the lipid uptake into the mucosa and/or reesterification to form triacylglycerols, but not the formation of chylomicrons, that is necessary for the stimulation of the secretion of alkaline phosphatase into the lymph.
IL-17 stimulates inflammatory responses via NF-κB and MAP kinase pathways in human colonic myofibroblasts
Authors: Hata Kazunori , Andoh Akira , Shimada Mitsue , Fujino Sanae , Bamba Shigeki , Araki Yoshio , Okuno Takafumi , Fujiyama Yoshihide , Bamba Tadao
Published: March 2015

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-κB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-κB activation within 45 min after stimulation. A blockade of NF-κB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1β or IL-17 + tumor necrosis factor (TNF)-α enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-α on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.
Estradiol-17β-D-glucuronide induces endocytic internalization of Bsep in rats
Authors: Crocenzi Fernando A. , Mottino Aldo D. , Cao Jingsong , Veggi Luis M. , Pozzi Enrique J. Sánchez , Vore Mary , Coleman Roger , Roma Marcelo G.
Published: March 2015

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17β-d-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 μmol/kg iv) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR-rats lacking Mrp2. DBcAMP (20 μmol/kg iv) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 μM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 μM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.
CCK-58 is the only detectable endocrine form of cholecystokinin in rat
Authors: Reeve Joseph R. , Green Gary M. , Chew Peter , Eysselein Viktor E. , Keire David A.
Published: March 2015

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 ± 0.5 and 17 ± 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.
Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP
Authors: Webster Cynthia R. L. , Blanch Christopher , Anwer M. Sawkat
Published: March 2015

cAMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Na+-taurocholate (TC) cotransporting peptide (NTCP) to the plasma membrane. Although translocation of NTCP may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate NTCP is, in part, dependent on cAMP-mediated increases in cytosolic Ca2+concentration ([Ca2+]), indicating that a Ca2+/calmodulin-dependent protein phosphatase (PP2B) may be involved. Thus we studied the role of PP2B using the inhibitor cypermethrin (CM). Freshly isolated hepatocytes were pretreated with 1–5 nM CM for 30 min followed by 15 min incubation with 10 μM 8-(4-chlorophenylthio)cAMP. CM (5 nM) and FK-506 (5 μM) inhibited cAMP-stimulated TC uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated NTCP dephosphorylation and translocation to 80 and 15% of the basal level, respectively. cAMP stimulated PP2B activity by 60%, and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated NTCP immunoprecipitated from control but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca2+] or decreases in mitogen-activated protein kinase (extracellular regulated kinases 1 and 2) activity. Taken together, these results suggest that cAMP dephosphorylates NTCP by activating PP2B in hepatocytes, and PP2B-mediated dephosphorylation of NTCP may be involved in cAMP-mediated NTCP translocation to the plasma membrane.
Direct biomechanical induction of endogenous calcineurin inhibitor Down Syndrome Critical Region-1 in cardiac myocytes
Authors: Wang Yanlin , De Keulenaer Gilles W. , Weinberg Ellen O. , Muangman Suphi , Gualberto Antonio , Landschulz Katherine T. , Turi Thomas G. , Thompson John F. , Lee Richard T.
Published: March 2015

Signaling through the protein phosphatase calcineurin may play a critical role in cardiac hypertrophy. The gene for Down Syndrome Critical Region-1 (DSCR1) encodes a protein that is an endogenous calcineurin inhibitor. This study was designed to test the hypothesis that DSCR1 is directly induced by biomechanical stimuli. Neonatal rat cardiac myocytes were exposed to biaxial cyclic mechanical strain; mechanical strain upregulated DSCR1 mRNA expression in a time- and amplitude-dependent manner (3.4 ± 0.2-fold at 8% strain for 6 h, n = 11, P < 0.01), and this induction was angiotensin II and endothelin I independent. Biomechanical induction of DSCR1 mRNA was partially blocked by calcineurin inhibition with cyclosporine A (30 ± 5%, n = 3, P < 0.01). DSCR1 promoter-reporter experiments showed that mechanical strain induced DSCR1 promoter activity by 2.3-fold and that this induction was completely inhibited by cyclosporin A. Furthermore, DSCR1 gene expression was increased in the left ventricles of mice with pressure-overload hypertrophy induced by transverse aortic banding. These data demonstrate that biomechanical strain directly induces gene expression for the calcineurin inhibitor DSCR1 in cardiac myocytes, indicating that mechanically induced DSCR1 may regulate the hypertrophic response to mechanical overload.
H2S-induced vasorelaxation and underlying cellular and molecular mechanisms
Authors: Zhao Weimin , Wang Rui
Published: March 2015

H2S is endogenously generated in vascular smooth muscle cells. The signal transduction pathways involved in the vascular effects of H2S have been unclear and were investigated in the present study. H2S induced a concentration-dependent relaxation of rat aortic tissues that was not affected by vascular denervation. The vasorelaxant potency of H2S was attenuated by the removal of the endothelium. Similarly, the blockade of nitric oxide synthase or the coapplication of the Ca2+-dependent K+channel blockers apamin and charybdotoxin reduced the H2S-induced relaxation of the endothelium-intact aortic tissues. Sodium nitroprusside (SNP)-induced relaxation was completely abolished by either 1 H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or NS- 2028, two soluble guanylate cyclase inhibitors. Instead of inhibition, ODQ and NS-2028 potentiated the H2S-induced vasorelaxation, which was suppressed by superoxide dismutase. The vasorelaxant effect of H2S was also significantly attenuated when Ca2+-free bath solution was used. Finally, pretreatment of aortic tissues with H2S reduced the relaxant response of vascular tissues to SNP. Our results demonstrate that the vascular effect of H2S is partially mediated by a functional endothelium and dependent on the extracellular calcium entry but independent of the activation of the cGMP pathway.
17β-Estradiol decreases vascular tone in cerebral arteries by shifting COX-dependent vasoconstriction to vasodilation
Authors: Ospina Jose A. , Duckles Sue P. , Krause Diana N.
Published: March 2015

We have previously shown that estrogen treatment increases cerebrovascular cyclooxygenase-1, prostacyclin synthase, and production of prostacyclin. Therefore, vascular tone and prostanoid production were measured to investigate functional consequences of estrogen exposure. Middle cerebral arteries were isolated from ovariectomized female Fischer-344 rats with or without chronic in vivo 17β-estradiol treatment. In vivo 17β-estradiol treatment increased cerebral artery diameter; functional endothelium was required for expression of these differences. The nonspecific cyclooxygenase inhibitor indomethacin constricted, whereas arachidonic acid dilated, cerebral arteries from estrogen-treated animals. Estrogen exposure increased production of prostacyclin by cerebral arteries. Conversely, in estrogen-deficient animals, indomethacin dilated and arachidonic acid constricted cerebral blood vessels. This correlated with vasorelaxation following inhibition of the thromboxane-endoperoxide receptor with SQ-29548 but not after selective blockade of thromboxane synthase with furegrelate, suggesting prostaglandin endoperoxide (i.e., PGH2) activity. Removal of the endothelium or selective blockade of cyclooxygenase-1 with SC-560 abolished estrogen-mediated differences in the effects of arachidonate on vessel diameter and on prostacyclin production by cerebral arteries. These data suggest 17β-estradiol decreases cerebrovascular tone by shifting the primary end product of the endothelial cyclooxygenase-1 pathway from the constrictor prostaglandin PGH2 to the vasodilator prostacyclin. These effects of estrogen may contribute to the heightened thromboresistance and enhanced cerebral blood flow documented in preversus postmenopausal women.
Load-dependent induction of apoptosis in multicellular myocardial preparations
Authors: Janssen P. M. L. , Hasenfuss G. , Zeitz O. , Lehnart S. E. , Prestle J. , Darmer D. , Holtz J. , Schumann H.
Published: March 2015

10.1152/ajpheart.00024.2001.—Increased mechanical load has been proposed as an inductor of apoptosis, but it is unknown whether this can occur in the range of pre- and afterloads that prevail in the beating heart. We investigated apoptosis in cultured rabbit multicellular myocardial preparations over several days. Muscles contracted in absence of pre- and afterload (unloaded isotonic), in absence of preload but in presence of afterload (unloaded isometric), or in presence of both (loaded isometric). After up to 48 h of continuous contractions, apoptosis was assessed by TdT-mediated nick-end labeling (TUNEL) assay and DNA ladder analysis. In muscles that contracted loaded isometric, apoptosis was detected after 6–24 h. After 48 h, apoptosis was most prominent in this group, reflected by a high level of DNA ladder intensity (DLI; 27.8 ± 11.5), whereas Bcl-xL (on RNA level) was significantly downregulated, and Fas remained unchanged. In unloaded isometric preparations, apoptosis was significantly less (6.9 ± 5.9 DLI) and very similar to those contracting unloaded isotonic (6.1 ± 5.1 DLI). We conclude that load-dependent apoptosis can occur at sarcomere lengths achievable in vivo and may mainly result from increased preload.
Mechanisms by which opening the mitochondrial ATP- sensitive K+channel protects the ischemic heart
Authors: Dos Santos Pierre , Kowaltowski Alicia J. , Laclau Muriel N. , Seetharaman Subramanian , Paucek Petr , Boudina Sihem , Thambo Jean-Benoit , Tariosse Liliane , Garlid Keith D.
Published: March 2015

Diazoxide opening of the mitochondrial ATP-sensitive K+(mitoKATP) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoKATPchannel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoKATPchannel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoKATPchannel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.