bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the cerebral cortical representation of heartburn in GERD patients
Authors: Kern Mark , Hofmann Candy , Hyde James , Shaker Reza
Published: January 2004

Although symptoms arising from the esophagus such as heartburn and pain can at times become challenging clinical problems, esophageal viscerosensation, especially with regard to chemical stimulation in humans, is incompletely understood. Our aims were 1) to characterize and ascertain the reproducibility of cerebral cortical registration of heartburn and 2) to elucidate the differences between these findings and those of esophageal subliminal acid stimulation in asymptomatic controls. We studied 11 gastroesophageal reflux disease (GERD) patients (9 males, 30–55 yr) and 15 healthy controls (8 males, 21–49 yr). Cerebral cortical functional magnetic resonance imaging (fMRI) activity was recorded twice in each subject, during two 5-min intervals of 0.1 N HCl, separated by 5 min of NaCl perfusion. Time from onset of acid perfusion to instant of fMRI signal increase and first report of heartburn averaged 1.60 ± 0.80 and 1.85 ± 0.60 min, respectively. Average maximum percent signal increase in the GERD patients (16.3 ± 3.5%) was significantly greater than that of healthy controls (3.8 ± 0.9%; P < 0.01). Temporal fMRI signal characteristics during heartburn were significantly different from those of subliminal acid stimulation in controls ( P < 0.01). Activated cortical regions included sensory/motor, parieto-occipital, cingulate and prefrontal regions, and the insula. There was 92% concordance between the activated Brodmann areas in repeated studies of GERD patients. Cortical activity associated with perceived and unperceived esophageal acid exposure in GERD patients and healthy controls, respectively, involves multiple brain regions but occurs more rapidly and with greater intensity in GERD patients than the activity in response to subliminal acid exposure in healthy controls. The cortical pain-processing pathway seems to be involved in perception of esophageal acid exposure and could explain the variations encountered in clinical practice defining this sensation.
Cerebral cortical representation of external anal sphincter contraction: effect of effort
Authors: Kern Mark K. , Arndorfer Ronald C. , Hyde James S. , Shaker Reza
Published: January 2004

The external anal sphincter (EAS) plays a critical role in maintaining fecal continence; however, cerebral cortical control of voluntary EAS contraction is not completely understood. Our aims were to determine the cortical areas associated with voluntary EAS contraction and to determine the effect of two levels of sphincter contraction effort on brain activity. Seventeen asymptomatic adults (ages 21-48, 9 male) were studied using functional magnetic resonance imaging (fMRI) to detect brain activity. Studies were done in two stages. In stage 1 (10 subjects, 5 male), anal sphincter pressure was monitored from a catheter-affixed bag. Subjects performed maximal and submaximal EAS contractions during two fMRI scanning sessions consisting of alternating 10-s intervals of sustained contraction and rest. In stage 2 studies, seven subjects (4 male) performed only maximum effort sphincter contractions without a catheter. EAS contraction was associated with multifocal fMRI activity in sensory/motor, anterior cingulate, prefrontal, parietal, occipital, and insular regions. Total cortical activity volume was significantly larger ( P < 0.05) for maximal (5,175 ± 720 μl) compared with submaximal effort contractions (2,558 ± 306 μl). Similarly, percent fMRI signal change was significantly higher ( P < 0.05) for maximal (4.8 ± 0.1%) compared with submaximal effort contractions (2.2 ± 0.1%). Cortical region-of-interest analysis showed the incidence of insular activation to be more common in women compared with men. Other cortical regions showed no such gender differences. fMRI activity detected in stage 2 showed similar regions of cortical activation to those of the stage 1 study. Willful contraction of the EAS is associated with multifocal cerebral cortical activity. The volume and intensity of cerebral cortical activation is commensurate with the level of contractile effort.
Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Krüppel-like factor
Authors: Hinnebusch Brian F. , Siddique Aleem , Henderson J. Welles , Malo Madhu S. , Zhang Wenying , Athaide Christopher P. , Abedrapo Mario A. , Chen Xinming , Yang Vincent W. , Hodin Richard A.
Published: January 2004

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.
Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice
Authors: Ito Yoshiya , Abril Edward R. , Bethea Nancy W. , McCuskey Robert S.
Published: January 2004

Nitric oxide (NO) is suggested to play a role in liver injury elicited by acetaminophen (APAP). Hepatic microcirculatory dysfunction also is reported to contribute to the development of the injury. As a result, the role of NO in hepatic microcirculatory alterations in response to APAP was examined in mice by in vivo microscopy. A selective inducible NO synthase (iNOS) inhibitor,l- N6-(1-iminoethyl)-lysine (l-NIL), or a nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), was intraperitoneally administered to animals 10 min before APAP gavage. l-NIL suppressed raised alanine aminotransferase (ALT) values 6 h after APAP, whereas l-NAME increased those 1.7-fold. Increased ALT levels were associated with hepatic expression of iNOS. l-NIL, but not l-NAME, reduced the expression. APAP caused a reduction (20%) in the numbers of perfused sinusoids. l-NIL restored the sinusoidal perfusion, but l-NAME was ineffective. APAP increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space. l-NIL tended to minimize this infiltration, whereas l-NAME further enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell phagocytic activity. This activity in response to APAP was blunted by l-NIL, whereas l-NAME further elevated it. l-NIL suppressed APAP-induced decreases in hepatic glutathione levels. These results suggest that NO derived from iNOS contributes to APAP-induced parenchymal cell injury and hepatic microcirculatory disturbances. l-NIL exerts preventive effects on the liver injury partly by inhibiting APAP bioactivation. In contrast, NO derived from constitutive isoforms of NOS exerts a protective role in liver microcirculation against APAP intoxication and thereby minimizes liver injury.
A new model of pacing in the mouse intestine
Authors: Daniel E. E. , Boddy Geoffrey , Bong Alicia , Cho WooJung
Published: January 2004

A simple model of pacing in mouse intestine to longitudinal (LM) as well as circular muscle (CM) has been developed. Undissected segments of LM or CM from mouse ileum or jejunum were prepared to record contractions, nerve functions were inhibited, and regular spontaneous contractions were recorded. These had the properties expected of interstitial cells of Cajal (ICC) paced contractions: ileum slower than jejunum, inhibited but not abolished by nicardipine, reduced in frequency by cyclopiazonic acid, abolished by Ca2+-free media, and high temperature dependence (Q10∼2.6-3.2). Nicardipine significantly reduced the pacing frequency in LM and CM. Intestinal segments from W/WV mice had few irregular contractions in CM but had regular contractions in LM. Other differences were found between LM and CM that suggest that the control of pacing of LM differed from pacing of CM. Moreover, both LM and CM segments in wild-type and W/WV and after cyclopiazonic acid responded to electrical pacing (50 V/cm, 50 or 100 ms) at 1 pulse per second. Temperature <26°C inhibited electrically paced contractions in CM. These findings suggest that the current models of ICC pacing need to be modified to apply to intact segments of mouse intestine.
Prolonged interferon-γ exposure decreases ion transport, NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo
Authors: Bertelsen Lone S. , Eckmann Lars , Barrett Kim E.
Published: January 2004

IFN-γ is elevated in intestinal inflammation and alters barrier and transport functions in human colonic epithelial cell lines, but its effects on normal human small intestinal epithelium in vivo are poorly defined. We investigated effects of prolonged IFN-γ exposure on ion transport and expression of transporters by using human fetal small intestinal xenografts. Xenograft-bearing mice were injected with IFN-γ, and 24 h later xenografts were harvested and mounted in Ussing chambers. Baseline potential difference (PD) was not affected by IFN-γ treatment. However, conductance was enhanced and agonist-stimulated ion transport was decreased. IFN-γ also decreased expression of the Na+-K+-2Cl-cotransporter and the α-subunit of Na+-K+-ATPase compared with controls, whereas levels of the calcium-activated Cl-channel and CFTR were unaltered. Thus prolonged exposure to IFN-γ leads to decreased ion secretion due, in part, to decreased ion transporter levels. These findings demonstrate the implications of elevated IFN-γ levels in human small intestine and validate the human intestinal xenograft as a model to study chronic effects of physiologically relevant stimuli.
Increased iNOS activity is essential for hepatic epithelial tight junction dysfunction in endotoxemic mice
Authors: Han Xiaonan , Fink Mitchell P. , Uchiyama Takashi , Yang Runkuan , Delude Russell L.
Published: January 2004

We tested the hypothesis that increased production of nitric oxide (NO·) by inducible NO· synthase (iNOS) is a key factor responsible for alterations in the expression, localization, and function of key tight junction (TJ) proteins in mice challenged with lipopolysaccharide (LPS, endotoxin). Endotoxemia was associated with hepatobiliary epithelial barrier dysfunction, as evidenced by increased plasma-to-bile leakage of FITC-labeled dextran (relative molecular mass 40 kDa) and increased circulating levels of bile acids and conjugated bilirubin. Immunoblotting revealed decreased expression of zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin in liver after injection of C57Bl/6J mice with 2 mg/kg Escherichia coli 0111:B4 LPS. Nonidet P-40-insoluble (i.e., TJ-associated) occludin and ZO-1 were virtually undetectable 12 and 18 h after injecting LPS. Immunofluorescence microscopy also revealed deranged subcellular localization of ZO-1 and occludin in endotoxemic mice. Pharmacological inhibition of iNOS activity using l- N6-(1-iminoethyl)lysine (5 mg/kg) or genetic ablation of iNOS ameliorated LPS-induced changes in hepatobiliary barrier function, and these strategies partially preserved TJ protein expression and localization. Steady-state levels of occludin and ZO-3 transcripts decreased transiently after injecting LPS but returned toward normal by 12 and 24 h after induction of endotoxemia, respectively. These results support the view that iNOS-dependent NO· production is an important factor contributing to hepatobiliary epithelial barrier dysfunction resulting from systemic inflammation and suggest that iNOS induction may play a role in the development of cholestatic jaundice in patients with severe sepsis.
Role of PGE2on gallbladder muscle cytoprotection of guinea pigs
Authors: Xiao Zuo-Liang , Biancani Piero , Behar Jose
Published: January 2004

H2O2and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2and damage functions of membrane proteins except for PGE2receptors. The aim of this study was to examine whether the preserved PGE2actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2prevented the inhibition of H2O2or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2before H2O2and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2prevents the muscle cell damage caused by H2O2and TCDC due to the resistance of PGE2receptors to agonist-induced desensitization. The preservation of PGE2receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.
Immunomagnetic enrichment of interstitial cells of Cajal
Authors: Ördög Tamás , Redelman Doug , Horowitz Nancy N. , Sanders Kenton M.
Published: January 2004

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was ∼63-fold and ∼8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca2+or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.
Norepinephrine effects on identified neurons of the rat dorsal motor nucleus of the vagus
Authors: y Valenzuela Isabel Martinez-Peña , Rogers Richard C. , Hermann Gerlinda E. , Travagli R. Alberto
Published: January 2004

The dorsal motor nucleus of the vagus (DMV) receives more noradrenergic terminals than any other medullary nucleus; few studies, however, have examined the effects of norepinephrine (NE) on DMV neurons. Using whole cell recordings in thin slices, we determined the effects of NE on identified gastric-projecting DMV neurons. Twenty-five percent of DMV neurons were unresponsive to NE, whereas the remaining 75% responded to NE with either an excitation (49%), an inhibition (26%), or an inhibition followed by an excitation (4%). Antrum/pylorus- and corpus-projecting neurons responded to NE with a similar percentage of excitatory (49 and 59%, respectively) and inhibitory (20% for both groups) responses. A lower percentage of excitatory (37%) and a higher percentage of inhibitory (36%) responses were, however, observed in fundus-projecting neurons. In all groups, pretreatment with prazosin or phenylephrine antagonized or mimicked the NE-induced excitation, respectively. Pretreatment with yohimbine or UK-14304 antagonized or mimicked the NE-induced inhibition, respectively. These data suggest that NE depolarization is mediated by α1-adrenoceptors, whereas NE hyperpolarization is mediated by α2-adrenoceptors. In 16 neurons depolarized by NE, amplitude of the action potential afterhyperpolarization (AHP) and its kinetics of decay (τ) were significantly reduced vs. control. No differences were found on the amplitude and τ of AHP in neurons hyperpolarized by NE. Using immunohistochemical techniques, we found that the distribution of tyrosine hydroxylase fibers within the DMV was significantly different within the mediolateral extent of DMV; however, distribution of cells responding to NE did not show a specific pattern of localization.
Calcium source diversity in feline lower esophageal sphincter circular and sling muscle
Authors: Muinuddin Ahmad , Neshatian Leila , Gaisano Herbert Y. , Diamant Nicholas E.
Published: January 2004

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca2+used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca2+-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca2+channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca2+from Ins(1,4,5)P3-sensitive stores is important in tone generation in both muscles. In Ca2+-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca2+stores with CPA or inhibition of Ins(1,4,5)P3-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca2+sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca2+entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.
ET-1 and TNF-α in HPS: analysis in prehepatic portal hypertension and biliary and nonbiliary cirrhosis in rats
Authors: Luo Bao , Liu Lichuan , Tang Liping , Zhang Junlan , Ling Yiqun , Fallon Michael B.
Published: January 2004

Common bile duct ligation (CBDL) triggers a molecular cascade resulting in the hepatopulmonary syndrome (HPS). Both increased hepatic endothelin-1 (ET-1) production and pulmonary vascular ETBreceptor expression with stimulation of endothelial nitric oxide synthase and TNF-α mediated inducible nitric oxide synthase and heme oxygenase-1 expression in pulmonary intravascular macrophages occur. Whether biliary cirrhosis is unique in triggering ET-1 and TNF-α alterations and HPS is unknown. We evaluated for HPS in rat prehepatic portal hypertension [partial portal vein ligation (PVL)], biliary (CBDL) and nonbiliary [thioacetamide treatment (TAA)] cirrhosis, and assessed ET-1 infusion in normal and PVL animals. Control, PVL, CBDL, TAA-treated, and ET-1-infused PVL animals had ET-1 and TNF-α levels measured and underwent molecular and physiological evaluation for HPS. HPS developed only in biliary cirrhosis in association with increased plasma ET-1 and TNF-α levels and the development of established molecular changes in the pulmonary microvasculature. In contrast, PVL did not increase ET-1 or TNF-α levels and TAA treatment increased TNF-α levels alone, and neither resulted in the full development of molecular or physiological changes of HPS despite portal pressure increases similar to those after CBDL. Exogenous ET-1 increased TNF-α levels and triggered HPS after PVL. Combination of ET-1 and TNF-α overproduction is unique to biliary cirrhosis and associated with experimental HPS. ET-1 infusion increases TNF-α levels and triggers HPS in prehepatic portal hypertension. ET-1 and TNF-α interact to trigger pulmonary microvascular changes in experimental HPS.
Apoptosis versus necrosis in acute pancreatitis
Authors: Bhatia Madhav
Published: January 2004

Acute pancreatitis is a disease of variable severity in which some patients experience mild, self-limited attacks, whereas others manifest a severe, highly morbid, and frequently lethal attack. The events that regulate the severity of acute pancreatitis are, for the most part, unknown. It is generally believed that the earliest events in acute pancreatitis occur within acinar cells and result in acinar cell injury. Other processes, such as recruitment of inflammatory cells and generation of inflammatory mediators, are believed to occur subsequent to acinar cell injury, and these “downstream” events are believed to influence the severity of the disease. Several recently reported studies, however, have suggested that the acinar cell response to injury may, itself, be an important determinant of disease severity. In these studies, mild acute pancreatitis was found to be associated with extensive apoptotic acinar cell death, whereas severe acute pancreatitis was found to involve extensive acinar cell necrosis but very little acinar cell apoptosis. These observations led to the hypothesis that apoptosis could be a favorable response to acinar cells and that interventions that favor induction of apoptotic, as opposed to necrotic, acinar cell death might reduce the severity of an attack of acute pancreatitis. Indeed, in an experimental setting, the induction of pancreatic acinar cell apoptosis protects mice against acute pancreatitis. Little is known about the mechanism of apoptosis in the pancreatic acinar cell, although some early attempts have been made in that direction. Also, clinical relevance of these experimental studies remains to be investigated.
Paradoxical roles of different nitric oxide synthase isoforms in colonic injury
Authors: Beck P. L. , Xavier R. , Wong J. , Ezedi I. , Mashimo H. , Mizoguchi A. , Mizoguchi E. , Bhan A. K. , Podolsky D. K.
Published: January 2004

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.
Tyrphostin AG 126 inhibits development of postoperative ileus induced by surgical manipulation of murine colon
Authors: Moore Beverley A. , Türler Andreas , Pezzone Michael A. , Dyer Kevin , Grandis Jennifer , Bauer Anthony J.
Published: January 2004

Manipulation of the bowel during abdominal surgery leads to a period of ileus, which is most severely manifested after procedures that directly involve the colon. Ileus is associated with the increased expression of proinflammatory cytokines and chemokines, a leukocytic infiltration into the muscularis, and the release of mediators from resident and infiltrating leukocytes that directly inhibit intestinal smooth muscle contractility. Phosphorylation of tyrosine residues on regulatory proteins by protein tyrosine kinases (PTKs) occurs at multiple steps in the signaling cascades that regulate the expression of proinflammatory genes. The purpose of this study was to determine whether inhibition of PTK activity will attenuate the inflammatory response associated with colonic ileus and lead to improved function. Using a rodent model of colonic postoperative ileus, we demonstrate that a single bolus injection of the PTK inhibitor tyrphostin AG 126 (15 mg/kg sc) before surgery significantly attenuates the surgically induced impairment of colonic contractility both in vivo and in vitro. Improvement in function was associated with a reduction in magnitude of inflammatory cell infiltrate and with a decrease in transcription of genes encoding proinflammatory mediators IL-1β and monocyte chemoattractant protein (MCP)-1, inducible nitric oxide synthase, and cyclooxygenase-2. Furthermore, tyrphostin AG 126 pretreatment significantly inhibited activation of multifactorial transcription factor NF-κB, which could form the basis for reduction in proinflammatory mediator expression. These data demonstrate for the first time that inhibition of PTK activity may represent a novel approach for management of ileus in the clinical setting.
C-type natriuretic peptide induces human colonic myofibroblast relaxation
Authors: Chitapanarux Taned , Chen Stephen L. , Lee Helen , Melton Andrew C. , Yee Hal F.
Published: January 2004

Intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. Our aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic Ca2+concentration. CNP induced relaxation of SEM within 10 s. By 10 min, relaxation reached a plateau that was sustained for 2 h. CNP-induced relaxation was saturable, with a maximal decrease in tension (51.7 ± 3.8 dyn) observed at 250 nM. SEM relaxation in response to CNP constituted ∼23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. Effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 min of CNP exposure and was sustained for at least 45 min. CNP also stimulated a large transient increase in cytosolic Ca2+concentration that occurred within 30 s and was nearly complete by 1 min. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP plays a role in intestinal wound healing.
Protection from ischemic liver injury by activation of A2Aadenosine receptors during reperfusion: inhibition of chemokine induction
Authors: Day Yuan-Ji , Marshall Melissa A. , Huang Liping , McDuffie Marcia J. , Okusa Mark D. , Linden Joel
Published: January 2004

Ischemia-reperfusion (I/R) injury occurs as a result of restoring blood flow to previously hypoperfused vessels or after tissue transplantation and is characterized by inflammation and microvascular occlusion. We report here that 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e), a selective agonist of the A2Aadenosine receptor (A2AAR), profoundly protects mouse liver from I/R injury when administered at the time of reperfusion, and protection is blocked by the antagonist ZM241385. ATL146e lowers liver damage by 90% as assessed by serum glutamyl pyruvic transaminase and reduces hepatic edema and MPO. Most protection remains if ATL146e treatment is delayed for 1 h but disappears when delayed for 4 h after the start of reperfusion. In mice lacking the A2AAR gene, protection by ATL1465e is lost and ischemic injury of short duration is exacerbated compared with wild-type mice, suggesting a protective role for endogenous adenosine. I/R injury causes induction of hepatic transcripts for IL-1α, IL-1β, IL-1Ra, IL-6, IL-10, IL-18, INF-β, INF-γ, regulated on activation, normal T cell expressed, and presumably secreted (RANTES), major intrinsic protein (MIP)-1α, MIP-2, IFN-γ-inducible protein (IP)-10, and monocyte chemotactic protein (MCP)-1 that are suppressed by administering ATL146e to wild-type but not to A2AAR knockout mice. RANTES, MCP-1, and IP-10 are notable as induced chemokines that are chemotactic to T lymphocytes. The induction of cytokines may contribute to transient lymphopenia and neutrophilia that occur after liver I/R injury. We conclude that most damage after hepatic ischemia occurs during reperfusion and can be blocked by A2AAR activation. We speculate that inhibition of chemokine and cytokine production limits inflammation and contributes to tissue protection by the A2AAR agonist ATL146e.
Induced nitric oxide promotes intestinal inflammation following hemorrhagic shock
Authors: Hierholzer Christian , Kalff Jörg C. , Billiar Timothy R. , Bauer Anthony J. , Tweardy David J. , Harbrecht Brian G.
Published: January 2004

In hemorrhagic shock (HS), increased cytokine production contributes to tissue inflammation and injury through the recruitment of neutrophils [polymorphonuclear cells (PMN)]. HS stimulates the early expression of inducible nitric oxide synthase (iNOS) that modulates proinflammatory activation after hemorrhage. Experiments were performed to determine the contribution of iNOS to gut inflammation and dysmotility after HS. Rats subjected to HS (mean arterial pressure 40 mmHg for 2.5 h followed by resuscitation and death at 4 h) demonstrated histological signs of mucosal injury, impairment of intestinal smooth muscle contractility, extravasation of PMN, and increased gut mRNA levels of ICAM-1, IL-6, and granulocyte colony-stimulating factor (G-CSF). In addition, DNA binding activity of NF-κB and Stat3, an IL-6 signaling intermediate, was significantly increased. In shocked rats treated with the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine at the time of resuscitation, histological signs of intestinal injury and PMN infiltration were reduced and muscle contractility was almost completely restored. Selective iNOS inhibition in shocked animals reduced the binding activity of NF-κB and Stat3 and reduced mRNA levels of ICAM-1, IL-6, and G-CSF. The results of studies using iNOS knockout mice subjected to HS were similar. We propose that early upregulation of iNOS contributes to the inflammatory response in the gut wall and participates in the activation of signaling cascades and cytokine expression that regulate intestinal injury, PMN recruitment, and impaired gut motility.
TNFR1 mediates the radioprotective effects of lipopolysaccharide in the mouse intestine
Authors: Riehl Terrence E. , Newberry Rodney D. , Lorenz Robin G. , Stenson William F.
Published: January 2004

LPS is radioprotective in the mouse small intestine through a mechanism that includes the synthesis of cyclooxygenase-2 (COX-2) and PGE2. The goal of this study was to identify the intermediate steps in this process. We used wild-type (WT) C57BL/6 mice and knockouts for tumor necrosis factor receptors 1 and 2 (TNFR1-/-, TNFR2-/-) and recombination-activating gene 1-/-mice. Mice were given parenteral LPS and then subjected to 12 Gy total body gamma irradiation. The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. Crypt cell apoptosis was assessed by histology. Parenteral administration of LPS induced COX-2 expression, PGE2production, and radioprotection in WT and TNFR2-/-mice but not in TNFR1-/-mice. TNFR1-/-mice were radioprotected by administration of exogenous 16,16-dimethyl PGE2. Immunohistochemical studies localized TNFR1 and COX-2 expression to subeptihelial fibroblasts and villus epithelial cells. Radiation-induced apoptosis was reduced by pretreatment with LPS in WT and TNFR2-/-mice but not in TNFR1-/-mice. In the absence of LPS, crypt survival was elevated in TNFR1-/-when compared with WT mice. These findings demonstrate that TNFR1 function is required for LPS-induced radioprotection in C57BL/6 mice and define an essential role for TNFR1 function in the induction of COX-2 expression and PGE2production in this process. The immunolocalization of TNFR1 and COX-2 expression to subepithelial fibroblasts following LPS administration suggests that this cell type plays an intermediate role in LPS-induced radioprotection in the intestine.
Mild irritant prevents ethanol-induced gastric mucosal microcirculatory disturbances through actions of calcitonin gene-related peptide and PGI2in rats
Authors: Saeki Takeo , Ohno Takashi , Kamata Kazuhisa , Arai Katsuharu , Mizuguchi Sumito , Katori Makoto , Saigenji Katsunori , Majima Masataka
Published: January 2004

Pretreatment with a mild irritant such as 1 M NaCl prevented ethanol-induced mucosal injury, which was abolished by indomethacin, suggesting involvement of endogenous PGs. With the use of intravital microscopy, we investigated the mechanism in microcirculation whereby a mild irritant prevents ethanol-induced mucosal injury. Microcirculation of the basal part of gastric mucosa in anesthetized rats was observed through a window with transillumination. Diameters of arterioles, collecting venules, and venules were measured with an electric microscaler. One molar NaCl alone caused dilation of arterioles and constrictions of collecting venules and venules, which were inhibited by indomethacin. Ethanol (50%) applied to mucosa constricted collecting venules and venules but dilated arterioles. Constriction of collecting venules resulted in mucosal congestion. Pretreatment with 1 M NaCl inhibited ethanol-induced constrictions of collecting venules and venules, and administration of indomethacin or a calcitonin gene-related peptide (CGRP) antagonist, CGRP-(8–37), abolished elimination of constrictions. Topical application (1 nM–10 μM) of PGE2or beraprost sodium (a PGI2analog) to microvasculature markedly and dose-dependently dilated arterioles, whereas that of PGE2, but not beraprost, slightly constricted collecting venules. Pretreatment of microvasculature with a nonvasoactive concentration of PGE2(100 nM) or beraprost (1 nM) completely inhibited ethanol-induced constriction of collecting venules. The inhibitory effect of beraprost but not of PGE2was abolished by CGRP-(8–37). Present results suggest that the mechanism whereby 1 M NaCl prevents ethanol-induced injury is elimination of constrictions of collecting venules and venules by CGRP whose release may be enhanced by PGI2but not by PGE2.
Responses of chronically hypoxic rat hearts to ischemia: KATP channel blockade does not abolish increased RV tolerance to ischemia
Authors: Forkel Joerg , Chen Xiaochao , Wandinger Susanne , Keser Florian , Duschin Alexey , Schwanke Uwe , Frede Stilla , Massoudy Parwis , Schulz Rainer , Jakob Heinz , Heusch Gerd
Published: January 2004

Chronic hypoxia may precondition the myocardium and protect from ischemia-reperfusion damage. We therefore examined the recovery of left and right ventricular function after ischemia and reperfusion (15 min each) in isolated blood-perfused working hearts from normoxic (Norm) and hypoxic (Hypo; 14 days, 10.5% O2) adult rats. In addition, the mRNA expression of hypoxia-inducible factor (HIF)-1α and the protein expression of endothelial nitric oxide synthase (eNOS) were measured. Postischemic left ventricular function recovered to 66 ± 6% and 67 ± 5% of baseline in Norm and Hypo, respectively. In contrast, postischemic right ventricular function was 93 ± 2% of baseline in Hypo vs. 67 ± 3% in Norm ( P < 0.05). Improved postischemic right ventricular function in Hypo (93 ± 2% and 96 ± 2% of baseline) was observed with 95% O2 or 21% O2 in the perfusate, and it was not attenuated by glibenclamide (5 and 10 μmol/l) (86 ± 4% and 106 ± 6% recovery). HIF-1α mRNA and eNOS protein expression were increased in both left and right hypoxic ventricles. In conclusion, postischemic right, but not left, ventricular function was improved by preceding chronic hypoxia. ATP-sensitive K+ channels are not responsible for the increased right ventricular tolerance to ischemia after chronic hypoxia in adult rat hearts.
Shock-induced arrhythmogenesis is enhanced by 2,3-butanedione monoxime compared with cytochalasin D
Authors: Cheng Yuanna , Li Li , Nikolski Vladimir , Wallick Don W. , Efimov Igor R.
Published: January 2004

Investigation of the mechanisms of arrhythmia genesis and maintenance has benefited from the use of optical mapping techniques that employ excitation-contraction uncouplers. We investigated the effects of the excitation-contraction uncouplers 2,3-butanedione monoxime (BDM) and cytochalasin D (Cyto D) on the induction and maintenance of arrhythmia by electric shocks. Electrical activity was optically mapped from anterior epicardium of rabbit hearts ( n = 9) during shocks (–100 V, 8 ms) applied from a ventricular lead at various phases of action potential duration (APD). Restitution curves were obtained using S1-S2 protocol and measurement of APD values at 70% of repolarization. Compared with Cyto D, BDM significantly shortened APD at 90% of repolarization, although no significant difference in dispersion of repolarization was observed. Wavelength was also shortened with BDM. In general, shock-induced arrhythmias with BDM and Cyto D were ventricular tachycardic in nature. With respect to shock-induced sustained arrhythmias, the vulnerable window was wider and the incidence was higher with BDM than with Cyto D. There was also a difference in the morphology of ventricular tachycardia (VT) between the two agents. The arrhythmias with BDM usually resembled monomorphic VT, especially those that lasted >30 s. In contrast, arrhythmias with Cyto D more resembled polymorphic VT. However, the average number of phase singularities increased under Cyto D vs. BDM, whereas no significant difference in the dominant frequency of shock-induced sustained arrhythmia was observed. BDM reduced the slope of the restitution curve compared with Cyto D, but duration of arrhythmia under BDM was significantly increased compared with Cyto D. In conclusion, BDM increased arrhythmia genesis and maintenance relative to Cyto D.
Polymorphism in gene coding for ACE determines different development of myocardial fibrosis in rats
Authors: Ocaranza María Paz , Díaz-Araya Guillermo , Carreño Juan E. , Muñoz David , Riveros Juan Pablo , Jalil Jorge E. , Lavandero Sergio
Published: January 2004

In humans, the effect of angiotensin-converting enzyme (ACE) gene polymorphisms in cardiovascular disease is still controversial. In the rat, a microsatellite marker in the ACE gene allows differentiation of the ACE gene polymorphism among strains with different ACE levels. We tested the hypothesis that this ACE gene polymorphism determines the extent of cardiac fibrosis induced by isoproterenol (Iso) in the rat. We used a male F2generation (homozygous LL and BB ACE genotypes determined by polymerase chain reaction) derived from two rat strains [Brown-Norway (BB) and Lewis (LL)] that differ with respect to their plasma ACE activities. For induction of left ventricular (LV) hypertrophy (LVH) and cardiac fibrosis, rats were infused with Iso (5 mg·kg–1·day–1) or saline (control) for 10 days and euthanized at day 1 after the last injection. The interstitial collagen volumetric fraction (ICVF), collagen I, and fibronectin content, but not collagen III content, were significantly higher in the homozygous BB rats than in homozygous LL rats. Differences in metalloprotease (MMP)-9, but not in MMP-2 activities as well as in cardiac cell proliferation, were also detected between LL and BB rats treated with Iso. LV ACE activity was higher in BB rats than LL rats and correlated with ICVF ( r = 0.61, P < 0.002). No changes were observed in plasma ACE activities, ANG II plasma or LV levels, plasma renin activity, and ACE and ANG II type 1 receptor (AT1R) mRNA levels in the LV of rats with the two different ACE polymorphisms. Iso induced a similar degree of LVH [assessed by an increase in LV weight 100 per body weight, LV-to-right ventricle (RV) ratio, and LV protein content] in LL and BB rats. We concluded that rats in the F2generation with high plasma ACE activity developed more fibrosis but to a similar degree of LVH compared with rats with low plasma ACE activity.
Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart
Authors: Zheng Meizi , Dilly Keith , Dos Santos Cruz Jader , Li Manxiang , Gu Yusu , Ursitti Jeanine A. , Chen Ju , Ross John , Chien Kenneth R. , Lederer Jonathan W. , Wang Yibin
Published: January 2004

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser16phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.
Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury
Authors: Jones Steven P. , Greer James J. M. , Kakkar Aman K. , Ware P. Derek , Turnage Richard H. , Hicks Michael , van Haperen Rien , de Crom Rini , Kawashima Seinosuke , Yokoyama Mitsuhiro , Lefer David J.
Published: January 2004

Previous studies indicate that deficiency of endothelial nitric oxide (NO) synthase (eNOS)-derived NO exacerbates myocardial reperfusion injury. We hypothesized that overexpression of eNOS would reduce the extent of myocardial ischemia-reperfusion (MI/R) injury. We investigated two distinct strains of transgenic (TG) mice overexpressing the eNOS gene (eNOS TG). Bovine eNOS was overexpressed in one strain (eNOS TG-Kobe), whereas the human eNOS gene was overexpressed in the other strain (eNOS TG-RT). Non-TG (NTG) and eNOS TG mice were subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion, and the extent of myocardial infarction was determined. Myocardial infarct size was reduced by 33% in the eNOS TG-Kobe strain ( P < 0.05 vs. NTG) and by 32% in the eNOS TG-RT strain ( P < 0.05 vs. NTG). However, postischemic cardiac function (cardiac output, fractional shortening) was not improved in the eNOS TG-Kobe mouse at 24 h of reperfusion [ P = not significant (NS) vs. NTG]. In additional studies, eNOS TG-Kobe mice were subjected to 30 min of myocardial infarction and 7 days of reperfusion. Fractional shortening and the first derivative of left ventricular pressure were measured in eNOS TG-Kobe and NTG mice, and no significant differences in contractility were observed ( P = NS) between the eNOS TG mice and NTG controls. Left ventricular end-diastolic pressure was significantly ( P < 0.05 vs. NTG) reduced in the eNOS TG-Kobe strain at 7 days of reperfusion. The cardioprotective effects of eNOS overexpression on myocardial infarct size were ablated by Nω-nitro-l-arginine methyl ester (300 mg/kg) pretreatment. Thus genetic overexpression of eNOS in mice attenuates myocardial infarction after MI/R but fails to significantly protect against postischemic myocardial contractile dysfunction in mice.