bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of ion secretion via proteinase-activated receptor-2 in human colon
Authors: Mall Marcus , Gonska Tanja , Thomas Jörg , Hirtz Stephanie , Schreiber Rainer , Kunzelmann Karl
Published: March 2015

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl− secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl− secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl−, by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca2+-ATPase inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-α may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.
Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury
Authors: Bhatia Madhav , Slavin John , Cao Yuqing , Basbaum Allan I. , Neoptolemos John P.
Published: March 2015

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury.
HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction
Authors: Bitar Khalil N.
Published: March 2015

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [32P]orthophosphate. C2-ceramide (0.1 μM), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 μM) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin.
Mathematical description of regenerative potentials recorded from circular smooth muscle of guinea pig antrum
Authors: Edwards F. R. , Hirst G. D. S.
Published: March 2015

Regenerative potentials evoked by intracellular current injection in single bundles of circular smooth muscle taken from guinea pig antrum have the characteristics of the secondary regenerative component of the slow wave occurring in the same muscle layer. Such regenerative depolarizations might result from a mechanism that responds to membrane polarization with a delayed increase in the rate of production of unitary potentials detected in this tissue. To test this possibility, a two-stage reaction leading to the formation of an intracellular messenger was proposed. The first forward reaction was voltage-dependent, in the manner described by the Hodgkin-Huxley transient Na conductance formalism, allowing simulation of anode break excitation, stimulus threshold strength-duration characteristics, and refractory behavior. A conventional dose-effect relationship was proposed to describe the dependence of the mean rate of discharge of unitary potentials on messenger concentration. Unitary potentials were modeled as unitary membrane conductance modulations with an empirically derived amplitude distribution and Poisson-distributed intervals. The model reproduces a range of spontaneous and evoked membrane potential changes characteristic of antral circular muscle bundles.
Role of TNF-α in gut mucosal changes after severe burn
Authors: Spies Marcus , Chappell Vicky L. , Dasu Mohan R. , Herndon David N. , Thompson James C. , Wolf Steven E.
Published: March 2015

Gut epithelial cell death by apoptosis is increased in the gut epithelium after severe burn associated with mucosal atrophy. We hypothesized that tumor necrosis factor (TNF)-α-TNF receptor (TNFR) interaction activates apoptosis in small bowel mucosal cells after severe burn. C57BL6 mice received a 30% total body surface area scald burn and were treated with neutralizing anti-TNF-α. The proximal small bowel was assessed for mucosal atrophy. Proliferation and apoptosis of mucosal cells were assessed by proliferative cell nuclear antigen-immunostaining and terminal deoxyuridine nick-end labeling assay, respectively. Mucosal height and mucosal cell number decreased after burn. Anti-TNF-α-treated mice showed significantly less mucosal atrophy. Proliferation of intestinal cells was not changed with burn or anti-TNF-α treatment. An over threefold increase in apoptotic cell number was seen after burn, which was diminished by anti-TNF-α treatment. Changes in gut mucosal homeostasis after severe burn are affected, in part, by the activation of apoptosis by TNF-α-TNFR interaction.
ERG K+ currents regulate pacemaker activity in ICC
Authors: Zhu Yaohui , Golden Catherine M. , Ye Jing , Wang Xuan-Yu , Akbarali Hamid I. , Huizinga Jan D.
Published: March 2015

Ether-à-go-go-related gene (ERG) K channels have been implicated in the generation of pacemaker activities in the heart. To study the presence and function of ERG K channels in the pacemaker cells of the small intestine [the interstitial cells of Cajal (ICC)], a combination of patch-clamp techniques, tissue and live cell immunohistochemistry, RT-PCR, and in vitro functional studies were performed. Nonenzymatically isolated ICC in culture were identified by vital staining and presence of rhythmic inward currents. RT-PCR showed the presence of ERG mRNA in the intestinal musculature, and immunohistochemistry on tissue and cultured cells demonstrated that protein similar to human ERG was concentrated on ICC in the Auerbach's plexus region. Whole cell ERG K+ currents were evoked on hyperpolarization from 0 mV (but not from -70 mV) up to -120 mV and showed strong inward rectification. The currents were inhibited by E-4031, cisapride, La3+, and Gd3+ but not by 50 μM Ba2+. The ERG K+ inward current had a typical transient component with fast activation and inactivation kinetics followed by significant steady-state current. E-4031 also inhibited tetraethylammonium (TEA)-insensitive outward current indicating that the ERG K+ current is operating at depolarizing potentials. In contrast to TEA, blockers of the ERG K+ currents caused marked increase in tissue excitability as reflected by an increase in slow-wave duration and an increase in superimposed action potential activity. In summary, ERG K channels in ICC contribute to the membrane potential and play a role in regulation of pacemaker activity of the small intestine.
EGF promotes gastric mucosal restitution by activating Na+/H+exchange of epithelial cells
Authors: Yanaka Akinori , Suzuki Hideo , Shibahara Takeshi , Matsui Hirofumi , Nakahara Akira , Tanaka Naomi
Published: March 2015

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na+/H+exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pHi) in SMC was measured fluorometrically, using 2′,7′- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pHiand enhanced restitution in the absence but not in the presence of serosal HCO[Formula: see text]. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na+/H+exchangers.
Sodium current in human intestinal interstitial cells of Cajal
Authors: Strege Peter R. , Ou Yijun , Sha Lei , Rich Adam , Gibbons Simon J. , Szurszewski Joseph H. , Sarr Michael G. , Farrugia Gianrico
Published: March 2015

Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of this study was to determine expression of a Na+current in human intestinal ICC and to determine the effects of the Na+current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c- kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A Na+current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-d-glucamine abolished and QX-314 (500 μM) blocked the Na+current, but nifedipine and Ni2+did not. The Na+current was activated by shear stress. Single-cell RT-PCR detected mRNA for the Na+α-subunit SCN5A in single human intestinal ICC. Lidocaine (200 μm) and QX-314 (500 μM) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive Na+channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function.
Interactions among the sevenHelicobacter pyloriproteins encoded by the urease gene cluster
Authors: Voland Petra , Weeks David L. , Marcus Elizabeth A. , Prinz Christian , Sachs George , Scott David
Published: March 2015

Survival of Helicobacter pylori in acid depends on intrabacterial urease. This urease is a Ni2+-containing oligomeric heterodimer. Regulation of its activity and assembly is important for gastric habitation by this neutralophile. The gene complex encodes catalytic subunits ( ureA/B), an acid-gated urea channel ( ureI), and accessory assembly proteins ( ureE–H). With the use of yeast two-hybrid analysis for determining protein-protein interactions, UreF as bait identified four interacting sequences encoding UreH, whereas UreG as bait detected five UreE sequences. These results were confirmed by coimmunoprecipitation and β-galactosidase assays. Native PAGE immunoblotting of H. pylori inner membranes showed interaction of UreA/B with UreI, whereas UreI deletion mutants lacked this protein interaction. Deletion of ureE–H did not affect this interaction with UreI. Hence, the accessory proteins UreE/G and UreF/H form dimeric complexes and UreA/B form a membrane complex with UreI, perhaps enabling assembly of the urease apoenzyme at the membrane surface and immediate urea access to intrabacterial urease to allow rapid periplasmic neutralization.
Role of epidermal growth factor and its receptor in chemotherapy-induced intestinal injury
Authors: Huang Frederick S. , Kemp Christopher J. , Williams Jodi L. , Erwin Christopher R. , Warner Brad W.
Published: March 2015

Several growth factors are trophic for the gastrointestinal tract and able to reduce the degree of intestinal damage caused by cytotoxic agents. However, studies of epidermal growth factor (EGF) for chemotherapy-induced intestinal injury are conflicting. The development of a transgenic mouse that specifically overexpresses EGF in the small intestine provided a unique opportunity to assess the contribution of EGF in mucositis. After a course of fluorouracil, transgenic mice fared no better than control mice. Weight recovery was inferior, and mucosal architecture was not preserved. Apoptosis was not decreased and proliferation was not increased in the crypts. To corroborate the findings in transgenic mice, ICR mice were treated with exogenous EGF after receiving fluorouracil. Despite ileal upregulation of native and activated EGF receptor, the mice were not protected from intestinal damage. No benefits were observed with different EGF doses or schedules or routes of EGF administration. Finally, mucositis was induced in mutant mice with specific defects of the EGF signaling axis. Compared with control mice, clinical and histological parameters of intestinal injury after fluorouracil were no different in waved-2 mice, which have functionally diminished EGF receptors, or waved-1 mice, which lack transforming growth factor-α, another major ligand for the EGF receptor. These findings do not support a critical role for EGF or its receptor in chemotherapy-induced intestinal injury.
Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice
Authors: Mang Christian F. , Truempler Sebastian , Erbelding Doris , Kilbinger Heinz
Published: March 2015

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N G-nitro-l-arginine (l-NNA) and the guanylyl cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of l-NNA were specifically antagonized byl-arginine. In contrast, l-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso- N-acetyl-dl-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.
Short fasting dramatically decreases rat duodenal secretory responsiveness to orexin A but not to VIP or melatonin
Authors: Flemström Gunnar , Sjöblom Markus , Jedstedt Gunilla , Åkerman Karl E. O.
Published: March 2015

Orexins are involved in the central nervous control of appetite and behavior, and in addition, they are present in endocrine cells and/or neurons in the intestine. The role of these peptides in peripheral regulation of intestinal secretion has not been investigated. We thus compared the effects of orexin A and some established secretagogues on duodenal HCO3- secretion in fed rats with effects in rats exposed to short (overnight) food deprivation. Rats were anesthetized with thiobarbiturate, a 12-mm segment of proximal duodenum with intact blood supply was cannulated in situ, and the alkaline secretion was titrated by pH stat. Secretagogues were supplied specifically to the duodenum by close intra-arterial infusion. Orexin A (60-600 pmol·kg-1·h-1) caused marked and dose-dependent stimulation of the duodenal secretion in fed animals but did not affect secretion in overnight food-deprived animals. Similarly, short fasting caused a 100-fold increase in the amount of the muscarinic agonist bethanechol (from 50 to 5,000 nmol·kg-1·h-1) required for stimulation of the secretion. In contrast, the secretory responses to VIP (50-1,000 pmol·kg-1·h-1) and melatonin (20-200 nmol·kg-1·h-1) were not affected. The appetite-regulating peptide orexin A is thus a stimulant of intestinal secretion, but the response to this peptide as well as the muscarinic agonist bethanechol is markedly dependent on previous intake of food. Overnight fasting is a standard experimental procedure in studies of gastrointestinal function and pathophysiology in humans and animals. Studies made on neuroendocrine control of intestinal secretion may require reevaluation with respect to feeding status.
The proximal colonic motor response to rectal mechanical and chemical stimulation
Authors: Bampton P. A. , Dinning P. G. , Kennedy M. L. , Lubowski D. Z. , Cook I. J.
Published: March 2015

We aimed to determine whether rectal distension and/or infusion of bile acids stimulates propagating or nonpropagating activity in the unprepared proximal colon in 10 healthy volunteers using a nasocolonic manometric catheter (16 recording sites at 7.5-cm spacing). Sensory thresholds and proximal colonic motor responses were assessed following rectal distension by balloon inflation and rectal instillation of chenodeoxycholic acid. Maximum tolerated balloon volume and the volume that stimulated a desire to defecate were both significantly ( P < 0.01) reduced after rectal chenodeoxycholic acid. The frequency of colonic propagating pressure wave sequences decreased significantly in response to initial balloon inflations ( P < 0.05), but the frequency doubled after subsequent chenodeoxycholic acid infusion ( P < 0.002). Nonpropagating activity decreased after balloon inflation, was not influenced by acid infusion, and demonstrated a further decrease in response to repeat balloon inflation. We concluded that rectal chenodeoxycholic acid in physiological concentrations is a potent stimulus for propagating pressure waves arising in the proximal colon and reduces rectal sensory thresholds. Rectal distension inhibits all colonic motor activity.
Glycine blocks the increase in intracellular free Ca2+due to vasoactive mediators in hepatic parenchymal cells
Authors: Qu Wei , Ikejima Kenichi , Zhong Zhi , Waalkes Michael P. , Thurman Ronald G.
Published: March 2015

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE2, which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca2+concentration ([Ca2+]i) in hepatic parenchymal cells by agonists released during stress, such as with PGE2and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca2+]iin individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE2caused a dose-dependent increase in [Ca2+]ifrom basal values of 130 ± 10 to maximal levels of 434 ± 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca2+dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca2+from intracellular stores, also partially blocked the increase in [Ca2+]icaused by PGE2, suggesting that intracellular Ca2+pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca2+pools are involved in the increase in [Ca2+]icaused by PGE2. Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca2+]idue to PGE2in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl−was omitted, glycine was much less effective in preventing the increase in [Ca2+]idue to PGE2. Phenylephrine, an α1-type adrenergic receptor agonist, also increased [Ca2+]i, as expected, from 159 ± 20 to 432 ± 43 nM. Glycine also blocked the increase in [Ca2+]idue to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca2+]iin hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.
The ferrireductase paraferritin contains divalent metal transporter as well as mobilferrin
Authors: Umbreit Jay N. , Conrad Marcel E. , Hainsworth Lucille N. , Simovich Marcia
Published: March 2015

Inorganic iron can be transported into cells in the absence of transferrin. Ferric iron enters cells utilizing an integrin-mobilferrin-paraferritin pathway, whereas ferrous iron uptake is facilitated by divalent metal transporter-1 (DMT-1). Immunoprecipitation studies using antimobilferrin antibody precipitated the previously described large-molecular-weight protein complex named paraferritin. It was previously shown that paraferritin functions as an intracellular ferrireductase, reducing ferric iron to ferrous iron utilizing NADPH as the energy source. It functions in the pathway for the cellular uptake of ferric iron. This multipeptide protein contains a number of active peptides, including the ferric iron binding protein mobilferrin and a flavin monooxygenase. The immunoprecipitates and purified preparations of paraferritin also contained DMT-1. This identifies DMT-1 as one of the peptides constituting the paraferritin complex. Since paraferritin functions to reduce newly transported ferric iron to ferrous iron and DMT-1 can transport ferrous iron, these findings suggest a role for DMT-1 in conveyance of iron from paraferritin to ferrochelatase, the enzyme utilizing ferrous iron for the synthesis of heme in the mitochondrion.
Intestinal lipoprotein assembly in apobec-1–/–mice reveals subtle alterations in triglyceride secretion coupled with a shift to larger lipoproteins
Authors: Xie Yan , Nassir Fatiha , Luo Jianyang , Buhman Kimberly , Davidson Nicholas O.
Published: March 2015

Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1–/–mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1–/–mice backcrossed into a C57BL/6 background. Inbred apobec-1–/–mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1–/–and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1–/–mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1–/–mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB.
5,6-Dichloro-ribifuranosylbenzimidazole- and apigenin-induced sensitization of colon cancer cells to TNF-α-mediated apoptosis
Authors: Farah Myriam , Parhar Kuljit , Moussavi Maryam , Eivemark Sharlene , Salh Baljinder
Published: March 2015

Tumor necrosis factor-α (TNF-α) is a multifunctional cytokine involved in the expression of many genes integral to the inflammatory response. In addition, it activates both apoptotic and survival pathways, the latter being mediated through the activation of the transcription factor nuclear factor-κB (NF-κB). Protein kinase CK2, a serine-threonine kinase that is universally upregulated in human malignancies, may be involved at multiple levels in this process. However, its role in mediating a survival response within colon cancer cells remains incompletely understood. Here we report that inhibition of CK2 in HCT-116 and HT-29 cells with the use of two specific CK2 inhibitors, 5,6-dichloro-ribifuranosylbenzimidazole (DRB) and apigenin, effected a synergistic reduction in cell survival when used in conjunction with TNF-α. Furthermore, there was a demonstrable synergistic reduction in colony formation in soft agar with the use of the same combinations. Western blot analysis showed that poly-ADP ribose polymerase and procaspase-3 cleavage complemented the fluorescence-activated cell sorter analysis findings of significantly increased subdiploid DNA-containing cell populations using these conditions. Remarkably, these events occurred in the absence of any reduction in the expression of the Bcl-2 family members Bcl-2, Mcl-1, and Bcl-xLor any change in the proapoptotic molecules Bad or Bax. One-hybrid NF-κB promoter assays utilizing a Gal4-p65 transactivation domain construct revealed that the TNF-induced transactivation was inhibited by both DRB and apigenin. This was associated with a concomitant reduction in the expression of a recognized anti-apoptotic NF-κB target, manganese superoxide dismutase, demonstrated by Q-PCR. Our findings indicate a potentially novel strategy for the treatment of colon cancer, one that targets CK2 simultaneous with TNF-α administration.
III. How is villin involved in the actin cytoskeleton dynamics in intestinal cells?
Authors: Athman Rafika , Louvard Daniel , Robine Sylvie
Published: March 2015

Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.
V. Polarized compartmentalization of antigen processing and Toll-like receptor signaling in intestinal epithelial cells
Authors: Hershberg Robert M.
Published: March 2015

The intestinal epithelial cell (IEC) is exposed at the apical surface to a high concentration of foreign antigen and bacterial products capable of triggering inflammatory responses. Complex intracellular pathways of antigen trafficking and the polarized expression of immunologically active receptors provide additional means to regulate the inflammatory pathways in these cells. In the case of human leukocyte antigen (HLA) class II heterodimers, surface expression is highly restricted to the basolateral surface, and this also appears to be the case for Toll-like receptor 5 (TLR5) on polarized T84 human colon cancer cells. Processing of soluble antigen via HLA class II in IEC can occur following internalization from the apical surface but is highly inefficient. In addition, certain bacteria can facilitate the transport of flagellin (the ligand for TLR5) across an intact epithelium. Disruption of the tight junctions between IECs, allowing direct access of antigen and flagellin to the basolateral surface of the cell, dramatically affects the functional outcome HLA class II and TLR5 pathways.
Effect of ligands that increase cAMP on caerulein-induced zymogen activation in pancreatic acini
Authors: Lu Z. , Kolodecik T. R. , Karne S. , Nyce M. , Gorelick F.
Published: March 2015

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 μM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.
Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes
Authors: Yoo James , Nichols Anthony , Song Jaekyung C. , Mammen Joshua , Calvo Isabel , Worrell Roger T. , Cuppoletti John , Matlin Karl , Matthews Jeffrey B.
Published: March 2015

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [3H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-ε. In addition, after bryostatin-1 treatment, PKC-δ and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.
Precursor peptide progastrin1-80reduces apoptosis of intestinal epithelial cells and upregulates cytochromecoxidase Vb levels and synthesis of ATP
Authors: Wu Hai , Owlia Azarmidokht , Singh Pomila
Published: March 2015

We recently reported that downregulation of gastrin gene expression in colon cancer cells significantly suppresses relative levels of mitochondrial cytochrome c (cyt c) oxidase Vb (Cox Vb) RNA and protein. These unexpected findings suggested the possibility that gastrin gene products [mainly progastrin (PG)] may be directly or indirectly mediating the observed effects in colon cancer cells. Because colon cancer cells do not respond to exogenous PG, we examined the possibility of whether PG regulates Cox Vb expression in gastrin-responsive intestinal epithelial cells (IECs) in vitro. Levels of Cox Vb RNA and protein were significantly increased in a dose-dependent manner in response to PG. Mitochondrial synthesis of ATP was also increased by approximately three- to fivefold in response to optimal concentrations (0.1-1.0 nm) of PG. Possible antiapoptotic effects of PG were additionally examined, because activation of caspases 9 and 3 had been noted in colon cancer cells downregulated for gastrin gene expression. We measured a significant loss in the levels of cyt c in the cytosol of PG-treated vs. control IEC cells, which correlated with a significant loss in the activation of caspases 9 and 3, resulting in a significant loss in DNA fragmentation on PG treatment of the cells. Our results thus suggest the novel possibility that the precursor PG peptide exerts direct antiapoptotic effects on IECs, which may contribute to the observed growth effects of PG on these cells. Additionally, Cox Vb gene appears to be an important intracellular target of PG, resulting in an increase in ATP levels, which may also contribute to the observed increase in the growth of target cells in response to PG.
Role of p55 tumor necrosis factor receptor 1 in acetaminophen-induced antioxidant defense
Authors: Chiu Hawjyh , Gardner Carol R. , Dambach Donna M. , Brittingham Jennie A. , Durham Stephen K. , Laskin Jeffrey D. , Laskin Debra L.
Published: March 2015

Tumor necrosis factor (TNF)-α is a macrophage-derived proinflammatory cytokine implicated in hepatotoxicity. In the present studies, p55 TNF receptor 1 (TNFR1) -/- mice were used to assess the role of TNF-α in acetaminophen-induced antioxidant defense. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increased serum alanine transaminases. This was correlated with a rapid depletion of hepatic glutathione (GSH). Whereas in WT mice GSH levels returned to control after 6–12 h, in TNFR1-/- mice recovery was delayed for 48 h. Delayed induction of heme oxygenase-1 and reduced expression of CuZn superoxide dismutase were also observed in TNFR1-/- compared with WT mice. This was associated with exaggerated hepatotoxicity. In WT mice, acetaminophen caused a time-dependent increase in activator protein-1 nuclear binding activity and in c-Jun expression. This response was significantly attenuated in TNFR1-/- mice. Constitutive NF-κB binding activity was detectable in livers of both WT and TNFR1-/- mice. A transient decrease in this activity was observed 3 h after acetaminophen in WT mice, followed by an increase that was maximal after 6–12 h. In contrast, in TNFR1-/- mice, acetaminophen-induced decreases in NF-κB activity were prolonged and did not return to control levels for 24 h. These data indicate that TNF-α signaling through TNFR1 plays an important role in regulating the expression of antioxidants in this model. Reduced generation of antioxidants may contribute to the increased sensitivity of TNFR1-/- mice to acetaminophen.
The splanchnic metabolism of flavonoids highly differed according to the nature of the compound
Authors: Crespy Vanessa , Morand Christine , Besson Catherine , Cotelle Nicole , Vézin Hervé , Demigné Christian , Rémésy Christian
Published: March 2015

The absorption and splanchnic metabolism of different flavonoids (namely quercetin, kaempferol, luteolin, eriodictyol, genistein, and catechin) were investigated in rats after an in situ perfusion of jejunum plus ileum (14 nmol/min). Net transfer across the brush border ranged widely according to the perfused compound (from 78% for kaempferol to 35% for catechin). This variation seems linked to the lipophilicity of a given flavonoid rather than to its three-dimensional structure. Except for catechin, conjugated forms of perfused flavonoids were also detected in the intestinal lumen, but the extent of this secretion depended on the nature of the perfused compounds (52% for quercetin to 11% for genistein). For some of the perfused aglycones, biliary secretion was an important excretion route: 30% of the perfused dose for genistein but only 1% for catechin. Thus the splanchnic metabolism of flavonoid is controlled by several factors: 1) the efficiency of their transfer through the brush border, 2) the intensity of the intestinal secretion of conjugates toward the mucosal and serosal sides, respectively, and 3) the biliary secretion of conjugates. These data suggested that the splanchnic metabolism of perfused flavonoids depends on the nature of the compound considered, which in turn influences their availability for peripheral tissues.
Epithelial Cells and Their Neighbors. III. Interactions between intraepithelial lymphocytes and neighboring epithelial cells
Authors: Havran Wendy L. , Jameson Julie M. , Witherden Deborah A.
Published: March 2015

Intraepithelial γδ-T cells are present in all epithelial tissues, where they reside in close contact with neighboring epithelial cells. Our data support the idea that the role of these cells is to monitor neighboring cells for signs of damage or disease. Once a problem is detected, the intraepithelial γδ-T cells can lyse damaged or malignant epithelial cells, directly participate in tissue repair through production of epithelial growth factors, and play a unique role in the recruitment of inflammatory cells to the site of damage. Intraepithelial γδ-T cells play unique roles in homeostasis and disease.
Basic fibroblast growth factor upregulates cyclooxygenase-2 in I407 cells through p38 MAP kinase
Authors: Tessner Teresa G. , Muhale Filipe , Schloemann Suzanne , Cohn Steven M. , Morrison Aubrey , Stenson William F.
Published: March 2015

The intestinal cell line I407 responds to basic fibroblast growth factor (bFGF) by upregulating cyclooxygenase-2 (COX-2) mRNA and protein expression and increasing PGE2production. bFGF treatment of I407 cells results in phosphorylation of p38, and the p38 inhibitor SB-203580 abrogates bFGF-induced PGE2 synthesis. Wild-type p38α (p38αWT) and dominant-negative p38α (p38αDN) stable transfectant clones of I407 cells were used to examine the role of the p38 MAP kinase pathway in the events controlling PGE2 synthesis after treatment with bFGF. Treatment of p38αWT clones with bFGF resulted in increased COX-2 protein levels and PGE2 synthesis similar to those seen in bFGF-treated control-transfected cells. In contrast, the p38αDN clones failed to upregulate COX-2 protein or increase PGE2 synthesis when treated with bFGF. Exogenous arachidonate did not restore PGE2 synthesis by p38αDN cells. bFGF treatment increased COX-2 mRNA stability, and the p38 inhibitor SB-203580 attenuated COX-2 mRNA stability in bFGF-treated I407 cells. These data demonstrate a crucial role for p38α in growth factor-induced PGE2 synthesis by intestinal cells. Furthermore, they indicate that p38 activity is required at a step distal to arachidonate release, most likely COX-2 upregulation, because exogenous arachidonate did not restore PGE2 synthesis.
Transfer of fatty acids between intracellular membranes: roles of soluble binding proteins, distance, and time
Authors: Weisiger R. A. , Zucker S. D.
Published: March 2015

Soluble fatty acid binding proteins (FABPs) are thought to facilitate exchange of fatty acids between intracellular membranes. Although many FABP variants have been described, they fall into two general classes. “Membrane-active” FABPs exchange fatty acids with membranes during transient collisions with the membrane surface, whereas “membrane-inactive” FABPs do not. We used modeling of fatty acid transport between two planar membranes to examine the hypothesis that these two classes catalyze different steps in intracellular fatty acid transport. In the absence of FABP, the steady-state flux of fatty acid from the donor to the acceptor membrane depends on membrane separation distance (d) approaching a maximum value ( Jmax) as d approaches zero. Jmaxis one-half the rate of dissociation of fatty acid from the donor membrane, indicating that newly dissociated fatty acid has a 50% chance of successfully reaching the acceptor membrane before rebinding to the donor membrane. For larger membrane separations, successful transfer becomes less likely as diffusional concentration gradients develop. The mean diffusional excursion of the fatty acid into the water phase (dm) defines this transition. For d≪dm, dissociation from the membrane is rate limiting, whereas for d≫dm, aqueous diffusion is rate limiting. All forms of FABP increase dmby reducing the rate of rebinding to the donor membrane, thus maintaining Jmaxover larger membrane separations. Membrane-active FABPs further increase Jmaxby catalyzing the rate of dissociation of fatty acids from the donor membrane, although frequent membrane interactions would be expected to reduce their diffusional mobility through a membrane-rich cytoplasm. Individual FABPs may have evolved to match the membrane separations and densities found in specific cell lines.
I. Morphology and topography of vagal afferents innervating the GI tract
Authors: Powley Terry L. , Phillips Robert J.
Published: March 2015

An understanding of the events initiating vago-vagal reflexes requires knowledge of mechanisms of transduction by vagal afferents. Such information presumes an understanding of receptor morphology and location. Anatomic studies have recently characterized two types of vagal afferents, both putative mechanoreceptors distributed in gastrointestinal (GI) smooth muscle. These two receptors are highly specialized in that they 1) are morphologically distinct, 2) have different smooth muscle targets, 3) form complexes with dissimilar accessory cells, and 4) vary in their regional distributions throughout the GI tract. By comparison, information on the architecture and regional distributions of other classes of vagal afferents, notably chemoreceptors, has only begun to accumulate. Progress on the study of the two mechanoreceptors, however, illustrates general principles and delineates experimental issues that may apply to other submodalities of vagal afferents. By extension from morphological and physiological observations on the two species of smooth muscle endings, it is reasonable to hypothesize that additional classes of vagal receptors are also differentiated morphologically and that they vary in structure, accessory cells, regional distributions, and other features. A full appreciation of vago-vagal reflexes will require thorough structural and regional analyses of each of the types of vagal receptors within the GI tract.
Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes
Authors: Cho Won Kyoo
Published: March 2015

Cell volume regulation plays a vital role in many cell functions. Recent study indicates that both K+ and Cl− channels are important for the regulatory volume decrease (RVD) of cholangiocarcinoma cells, but its physiological significance is unclear due to the tumorous nature of the cells used. This present study reports the RVD of normal mouse cholangiocytes by using freshly isolated bile duct cell clusters (BDCC). A relatively simple and practical method of measuring the cross-sectional area of BDCCs by quantitative videomicroscopy was used to indirectly measure their volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by 5-nitro-2′-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide, suggesting its dependence on certain chloride channels, such as volume-activated chloride channels. It is also inhibited by barium chloride but not by tetraethylammonium chloride, indicating its dependence on certain potassium channels. However, cAMP agonists had no significant effect on the RVD of BDCCs. This indirect method described can be used to study the RVD of cholangiocytes from normal as well as genetically altered mouse livers.
Ontogenetic expression and regulation of Na+-d-glucose cotransporter in jejunum of domestic chicken
Authors: Barfull Anna , Garriga Carles , Mitjans Montserrat , Planas Joana M.
Published: March 2015

To evaluate the effect of age on sugar transport, we determined the uptake of methyl α-d-glucopyranoside and the abundance of the Na+-d-glucose cotransporter (SGLT1) in jejunal brush-border membrane (BBM) vesicles of 2-day- and 5-wk-old chickens. Methyl α-d-glucopyranoside transport per BBM protein was 40% lower in adults than in newly hatched chickens. This finding was matched by parallel declines in site density of SGLT1, which were detected by Western blot. The immunohistochemical study showed that SGLT1 was exclusively located in the BBM of enterocytes along the entire villus and was absent in the crypt in both age groups, and there was an 11-fold increase in the total absorptive area during development. Northern blot studies of the abundance of SGLT1 mRNA showed similar levels for the groups studied. We conclude that the age-related decline in Na+-dependent hexose transport per unit of BBM protein in the chicken jejunum is due to a reduction in the density of SGLT1 cotransporter and is regulated by a posttranscriptional mechanism.