bluuebunny/crossref_metadata_2025 huggingface dataset:
I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation
Authors: Grisham Matthew B. , Jourd’Heuil David , Wink David A.
Published: December 2017

The role of nitric oxide (NO) in inflammation represents one of the most studied yet controversial subjects in physiology. A number of reports have demonstrated that NO possesses potent anti-inflammatory properties, whereas an equally impressive number of studies suggest that NO may promote inflammation-induced cell and tissue dysfunction. The reasons for these apparent paradoxical observations are not entirely clear; however, we propose that understanding the physiological chemistry of NO and its metabolites will provide a blueprint by which one may distinguish the regulatory/anti-inflammatory properties of NO from its deleterious/proinflammatory effects. The physiological chemistry of NO is complex and encompasses numerous potential reactions. In an attempt to simplify the understanding of this chemistry, the physiological aspects of NO chemistry may be categorized into direct and indirect effects. This type of classification allows for consideration of timing, location, and rate of production of NO and the relevant targets likely to be affected. Direct effects are those reactions in which NO interacts directly with a biological molecule or target and are thought to occur under normal physiological conditions when the rates of NO production are low. Generally, these types of reactions may serve regulatory and/or anti-inflammatory functions. Indirect effects, on the other hand, are those reactions mediated by NO-derived intermediates such as reactive nitrogen oxide species derived from the reaction of NO with oxygen or superoxide and are produced when fluxes of NO are enhanced. We postulate that these types of reactions may predominate during times of active inflammation. Consideration of the physiological chemistry of NO and its metabolites will hopefully allow one to identify which of the many NO-dependent reactions are important in modulating the inflammatory response and may help in the design of new therapeutic strategies for the treatment of inflammatory tissue injury.
Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells
Authors: Ikejima Kenichi , Enomoto Nobuyuki , Seabra Vitor , Ikejima Ayako , Brenner David A. , Thurman Ronald G.
Published: December 2017

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-α (TNF-α) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-α mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.
Diazepam-binding inhibitor33–50elicits Ca2+oscillation and CCK secretion in STC-1 cells via L-type Ca2+channels
Authors: Yoshida Hitoshi , Tsunoda Yasuhiro , Owyang Chung
Published: December 2017

We recently isolated and characterized 86-amino acid CCK-releasing peptide from porcine intestinal mucosa. The sequence of this peptide is identical to that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administration of DBI stimulates the CCK release and elicits pancreatic secretion in rats. In this study we utilized a murine tumor cell line (STC-1 cells) that contains CCK to examine if DBI directly acts on these cells to stimulate CCK release. We investigated the cellular mechanisms responsible for this action. We showed that DBI33–50, a biologically active fragment of DBI1–86, significantly stimulated CCK secretion in STC-1 cells. This action was abolished by Ca2+-free medium. The mean basal intracellular Ca2+concentration ([Ca2+]i) was 52 nM in fura 2-loaded STC-1 cells. DBI33–50(1–1,000 nM) elicited Ca2+oscillations; DBI33–50(10 nM) increased the oscillation frequency to 5 cycles/10 min and elicited a net [Ca2+]iincrease (peak − basal) to 157 nM. In contrast, bombesin and forskolin caused an initial transient [Ca2+]ifollowed by a small sustained [Ca2+]iplateau. Withdrawal of extracellular Ca2+abolished Ca2+oscillations stimulated by DBI33–50. L-type Ca2+channel blockers nifedipine and diltiazem (3–10 μM) markedly attenuated DBI-stimulated Ca2+oscillations. In other cell types L-type Ca2+channels are activated by cAMP-protein kinase A. DBI33–50failed to stimulate cAMP formation in STC-1 cells. Similarly, DBI33–50had no effect on myo-inositol 1,4,5-trisphosphate concentration ([IP3]), whereas bombesin caused an eightfold increase in [IP3] over basal. In addition, inhibitors of phospholipase C (U-73122), phospholipase A2(ONO-RS-082), and protein tyrosine kinase (genistein) did not alter the Ca2+oscillations elicited by DBI33–50. It appears that DBI33–50acts directly on STC-1 cells to elicit Ca2+oscillations via the voltage-dependent L-type Ca2+channels, resulting in the secretion of CCK. Mediation of this action is by intracellular mechanisms independent of the traditional signal transduction pathways, including phospholipase C, phospholipase A2, protein tyrosine kinase, and cAMP systems.
Mechanisms underlying the anti-inflammatory actions of central corticotropin-releasing factor
Authors: Casadevall Maria , Saperas Esteban , Panés Julián , Salas Azucena , Anderson Donald C. , Malagelada Juan R. , Piqué Josep M.
Published: December 2017

Immune activation of hypothalamic corticotropin-releasing factor (CRF) provides a negative feedback mechanism to modulate peripheral inflammatory responses. We investigated whether central CRF attenuates endothelial expression of intercellular adhesion molecule 1 (ICAM-1) and leukocyte recruitment during endotoxemia in rats and determined its mechanisms of action. As measured by intravital microscopy, lipopolysaccharide (LPS) induced a dose-dependent increase in leukocyte rolling, adhesion, and emigration in mesenteric venules, which was associated with upregulation of endothelial ICAM-1 expression. Intracisternal injection of CRF abrogated both the increased expression of ICAM-1 and leukocyte recruitment. Intravenous injection of the specific CRF receptor antagonist astressin did not modify leukocyte-endothelial cell interactions induced by a high dose of LPS but enhanced leukocyte adhesion induced by a low dose. Blockade of endogenous glucocorticoids but not α-melanocyte-stimulating hormone (α-MSH) receptors reversed the inhibitory action of CRF on leukocyte-endothelial cell interactions during endotoxemia. In conclusion, cerebral CRF blunts endothelial upregulation of ICAM-1 and attenuates the recruitment of leukocytes during endotoxemia. The anti-inflammatory effects of CRF are mediated by adrenocortical activation and additional mechanisms independent of α-MSH.
Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini
Authors: Saluja A. K. , Bhagat L. , Lee H. S. , Bhatia M. , Frossard J. L. , Steer M. L.
Published: December 2017

The mechanisms responsible for intrapancreatic digestive enzyme activation as well as the relationship between that activation and cell injury during pancreatitis are not understood. We have employed an in vitro system in which freshly prepared pancreatic acini are exposed to a supramaximally stimulating concentration of the CCK analog caerulein to explore these issues. We find that in vitro trypsinogen activation depends on the continued presence of Ca2+in the suspending medium and that it is half-maximal in the presence of 0.3 mM Ca2+. Caerulein-induced trypsinogen activation can be halted by removal of Ca2+from the suspending medium or by chelation of intracellular Ca2+. Increasing intracellular Ca2+with either ionomycin or thapsigargin does not induce trypsinogen activation. We have monitored cell injury by measuring the leakage of lactate dehydrogenase (LDH) from acini and by quantitating intercalation of propidium iodide (PI) into DNA. Leakage of LDH and intercalation of PI in response to supramaximal stimulation with caerulein can be detected only after caerulein-induced trypsinogen activation has already occurred, and these indications of cell injury can be prevented by addition of a cell-permeant protease inhibitor. Our findings indicate that caerulein-induced intra-acinar cell activation of trypsinogen depends on a rise in intracellular Ca2+, which reflects entry of Ca2+from the suspending medium. Intra-acinar cell activation of trypsinogen is an early as well as a critical event in pancreatitis. The subsequent cell injury in this model is mediated by activated proteases.
Intracisternal TRH analog increases gastrin release and corpus histidine decarboxylase activity in rats
Authors: Song Min , Yang Hong , Walsh John H. , Ohning Gordon , Wong Helen , Taché Yvette
Published: December 2017

Thyrotropin-releasing hormone (TRH) acts in brain stem nuclei to induce vagally mediated stimulation of gastric secretion. The effects of intracisternal injection of the TRH analog RX-77368 on plasma gastrin levels and corpus histidine decarboxylase (HDC) activity were studied in 48-h fasted conscious rats. RX-77368 (25–100 ng) increased plasma gastrin levels by threefold at 30 min, which remained significantly higher than control at 2 and 4 h postinjection. Corpus HDC activity began to increase at 2 h and reached a peak at 4 h postinjection with a 21-fold maximum response observed at 50 ng. Morphological changes in the appearance of corpus HDC-immunoreactive cells correlated well with HDC activity. Pretreatment with gastrin monoclonal antibody completely prevented RX-77368 stimulatory effects on HDC activity. Atropine significantly attenuated gastrin increase at 30 min by 26%. These results indicated that in conscious fasted rats, TRH analog acts in the brain to increase corpus HDC activity in the enterochromaffin-like cells, which involves gastrin release stimulated by central TRH analog.
Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in Caco-2 cells
Authors: Fleet James C. , Wood Richard J.
Published: December 2017

Calcium transport in the apical-to-basolateral (A-to-B) or B-to-A direction was examined in cells treated with 10 nM 1,25-dihydroxyvitamin D3[1,25(OH)2D3, calcitriol] for up to 72 h. Net A-to-B calcium transport was positive at all time points and increased from 0.14 ± 0.06 to 0.50 ± 0.01 nmol ⋅ well−1⋅ min−1after 72 h of calcitriol treatment. Neither phenol red transport nor transepithelial electrical resistance was altered by calcitriol treatment, suggesting that the increase in net A-to-B calcium transport was not due to paracellular movement. Neither 25-hydroxyvitamin D3nor 24,25-dihydroxyvitamin D3(100 nM, 48 h) alters basal or calcitriol-stimulated A-to-B calcium transport. Treatment with the calmodulin antagonist trifluoperazine (50 μM) reduced calcitriol-stimulated A-to-B Ca transport by 56%. The transcription inhibitor actinomycin D inhibited calcitriol-regulated A-to-B calcium transport as well as calbindin D9kand 24-hydroxylase mRNA accumulation. These data demonstrate that calcitriol-mediated A-to-B calcium transport in Caco-2 cells is a specific, transcellular process that requires transcriptional events normally mediated through the vitamin D receptor.
Induction of epithelial Na+channel in rat ileum after proctocolectomy
Authors: Koyama Kaori , Sasaki Iwao , Naito Hiroo , Funayama Yuji , Fukushima Kouhei , Unno Michiaki , Matsuno Seiki , Hayashi Hisayoshi , Suzuki Yuichi
Published: December 2017

In patients with colectomy, epithelial transport function in the remnant small intestine can be regulated in response to the increased fecal electrolyte and fluid loss. Using a rat colectomy model, we investigated the Na+and K+transport mechanisms underlying the intestinal response. Proctocolectomy with ileoanal anastomosis was performed on rats. The small intestinal mucosa was mounted in Ussing chambers; then short-circuit currents and22Na+fluxes were measured. mRNA expression of the epithelial Na+channel (ENaC) was determined by Northern blotting. Amiloride-sensitive, electrogenic Na+absorption appeared in the ileum after proctocolectomy. This functional change was accompanied by the chronological induction of mRNAs for α-, β-, and γ-subunits of the ENaC in the ileum. Tetraethylammonium-sensitive short-circuit current was also activated. We conclude that electrogenic Na+absorption and probably K+secretion are induced in the ileum after proctocolectomy. This induction of electrogenic Na+absorption is probably mediated by the increase in the mRNA levels for all three types of subunits of the ENaC and may contribute to the recovery from the increased fecal Na+loss.
Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide
Authors: Bilzer Manfred , Jaeschke Hartmut , Vollmar Angelika M. , Paumgartner Gustav , Gerbes Alexander L.
Published: December 2017

The generation of reactive oxygen species (ROS) by activated Kupffer cells contributes to liver injury following liver preservation, shock, or endotoxemia. Pharmacological interventions to protect liver cells against this inflammatory response of Kupffer cells have not yet been established. Atrial natriuretic peptide (ANP) protects the liver against ischemia-reperfusion injury, suggesting a possible modulation of Kupffer cell-mediated cytotoxicity. Therefore, we investigated the mechanism of cytoprotection by ANP during Kupffer cell activation in perfused rat livers of male Sprague-Dawley rats. Activation of Kupffer cells by zymosan (150 μg/ml) resulted in considerable cell damage, as assessed by the sinusoidal release of lactate dehydrogenase and purine nucleoside phosphorylase. Cell damage was almost completely prevented by superoxide dismutase (50 U/ml) and catalase (150 U/ml), indicating ROS-related liver injury. ANP (200 nM) reduced Kupffer cell-induced injury via the guanylyl cyclase-coupled A receptor (GCA receptor) and cGMP: mRNA expression of the GCA receptor was found in hepatocytes, endothelial cells, and Kupffer cells, and the cGMP analog 8-bromo-cGMP (8-BrcGMP; 50 μM) was as potent as ANP in protecting from zymosan-induced cell damage. ANP and 8-BrcGMP significantly attenuated the prolonged increase of hepatic vascular resistance when Kupffer cell activation occurred. Furthermore, both compounds reduced oxidative cell damage following infusion of H2O2(500 μM). In contrast, superoxide anion formation of isolated Kupffer cells was not affected by ANP and only moderately reduced by 8-BrcGMP. In conclusion, ANP protects the liver against Kupffer cell-related oxidant stress. This hormonal protection is mediated via the GCA receptor and cGMP, suggesting that the cGMP receptor plays a critical role in controlling oxidative cell damage. Thus ANP signaling should be considered as a new pharmacological target for protecting liver cells against the inflammatory response of activated Kupffer cells without eliminating the vital host defense function of these cells.
Effect of heat stress on rabbit esophageal epithelium
Authors: Tobey Nelia A. , Sikka Dipali , Marten Esteban , Caymaz-Bor Canan , Hosseini S. Seraj , Orlando Roy C.
Published: December 2017

Hot beverages expose the esophageal epithelium to temperatures as high as 58°C. To study the impact of such temperatures, rabbit esophageal epithelium was exposed to luminal heat or both luminal and serosal heat while mounted in Ussing chambers. Luminal heat, mimicking exposure to hot beverages, reduced potential difference (PD) and resistance ( R) when applied at ≥49°C and reduced short-circuit current ( I sc) at ≥60°C. At ≥60°C, subepithelial blisters developed. Higher temperatures reduced R only moderately and reversibly. In contrast, the I sc declined sharply and irreversibly once threshold was reached. Luminal and serosal heat also reduced PD, I sc, and R, although the threshold for reduction in I scwas now similar to that for R. Additionally, luminal and serosal heat reduced I sc more than R for any given temperature and resulted in blisters at lower temperatures (50°C) than luminal heat alone. The heat-induced decline in I sc was attributed in part to inactivation of Na-K-ATPase activity, although other transport systems could have been equally affected, and the decline in R to an increase in paracellular permeability. The latter effect on R also contributed to an increase in tissue sensitivity to luminal acid damage. Consumption of hot beverages exposes the esophagus to temperatures that can negatively impact epithelial structure and function. Impaired barrier function by heat increases the risk of esophageal damage by subsequent contact with (refluxed) gastric acid. These findings help explain in part the association between esophageal disease and consumption of hot beverages.
Roles of IL-1 and TNF in the decreased ileal muscle contractility induced by lipopolysaccharide
Authors: Lodato Robert F. , Khan A. Rizwan , Zembowicz Malgorzata J. , Weisbrodt Norman W. , Pressley Thomas A. , Li Yong-Fang , Lodato Joseph A. , Zembowicz Artur , Moody Frank G.
Published: December 2017

Gastrointestinal stasis during sepsis may be associated with gastrointestinal smooth muscle dysfunction. Endotoxin [lipopolysaccharide (LPS)] impairs smooth muscle contraction, in part through inducible nitric oxide synthase (NOS II) and enhanced nitric oxide production. We studied the roles of tumor necrosis factor-α (TNF) and interleukin-1 (IL-1) in this process by using TNF binding protein (TNFbp) and IL-1 receptor antagonist (IL-1ra). Rats were treated with TNFbp and IL-1ra, or their vehicles, 1 h before receiving LPS or saline. At 5 h after LPS, contractility was measured in strips of ileal longitudinal smooth muscle, and NOS II activity was measured in full-thickness segments of ileum. LPS decreased maximum stress (mean ± SE) from 508 ± 55 (control) to 355 ± 33 g/cm2( P < 0.05). Pretreatment with TNFbp plus IL-1ra prevented the LPS-induced decrease. Separate studies of TNFbp alone or IL-1ra alone indicated that, at the doses and timing used, TNFbp was more effective. LPS also increased NOS II activity by >10-fold ( P < 0.01) over control. This increase was prevented by TNFbp plus IL-1ra ( P = not significant vs. control). We conclude that the LPS-induced increase in NOS II activity and the decrease in ileal muscle contractility are mediated by TNF and IL-1.
Effects of substance P on human colonic mucosa in vitro
Authors: Riegler Martin , Castagliuolo Ignazio , So Peter T. C. , Lotz Margaret , Wang Chi , Wlk Michael , Sogukoglu Tacettin , Cosentini Enrico , Bischof Georg , Hamilton Gerhard , Teleky Bela , Wenzl Etienne , Matthews Jeffrey B. , Pothoulakis Charalabos
Published: December 2017

Previous studies indicated that the peptide substance P (SP) causes Cl−-dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10−8to 10−6M) induced a rapid, monophasic concentration and Cl−-dependent, bumetanide-sensitive short-circuit current ( Isc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1(SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced Iscincrease without impairing forskolin- and carbachol-mediated Iscincrease. We conclude that SP stimulates Cl−-dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.
Effect of diaspirin cross-linked hemoglobin on normal and postischemic microcirculation of the rat pancreas
Authors: von Dobschuetz Ernst , Hoffmann Tomas , Engelschalk Clemens , Messmer Konrad
Published: December 2017

Microcirculatory alterations with reduced nutritive supply to the pancreas could be the cause of hyperamylasemia, which occurs in some patients receiving the vasoactive oxygen carrier diaspirin cross-linked hemoglobin (DCLHb) in clinical studies. Therefore, the effects of DCLHb on rat pancreas microcirculation were evaluated. Anesthetized Sprague-Dawley rats received one of the following treatments during baseline conditions ( n = 7 rats/group): 10% hydroxyethyl starch (HAES) (0.4 ml/kg), DCLHb (400 mg/kg), or DCLHb (1,400 mg/kg). After 1 h of complete, reversible pancreatic ischemia, other animals received 10% HAES (0.4 ml/kg) or DCLHb (400 mg/kg) during the onset of reperfusion. The number of red blood cell-perfused capillaries (functional capillary density, FCD) and the level of leukocyte adherence in postcapillary venules in the pancreas were assessed by means of intravital microscopy during 2 h after treatment. In the nonischemic groups, FCD was 18% greater after DCLHb (1,400 mg/kg) than after 10% HAES treatment without any increase in leukocyte adherence. In the inschemia-reperfusion (I/R) 10% HAES group, FCD was significantly ( P < 0.05) lowered, leukocyte adherence enhanced, and mean arterial pressure (MAP) reduced by 31% compared with nonischemic animals. DCLHb treatment in the I/R group resulted in a slight increase in FCD, a significant ( P < 0.05) reduction of leukocyte adherence, and a complete restoration of MAP compared with the animals of the I/R control group. Thus our data provide no evidence for a detrimental effect on the pancreatic microcirculation or an enhanced risk of postischemic pancreatitis by DCLHb.
Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration
Authors: Liu Xingrong , LeCluyse Edward L. , Brouwer Kenneth R. , Gan Liang-Sheng L. , Lemasters John J. , Stieger Bruno , Meier Peter J. , Brouwer Kim L. R.
Published: December 2017

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.
Molecular diversity of KVα- and β-subunit expression in canine gastrointestinal smooth muscles
Authors: Epperson Anne , Bonner Helena P. , Ward Sean M. , Hatton William J. , Bradley Karri K. , Bradley Michael E. , Trimmer James S. , Horowitz Burton
Published: December 2017

Voltage-activated K+(KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the KV currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different KV channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of KV1.4, KV1.5, KV1.6, KV2.2, and KV4.3 transcripts in all regions of the GI tract examined. Transcripts encoding KV1.2, KVβ1.1, and KVβ1.2 subunits were differentially expressed. KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, KV4.1, KV4.2, and KVβ2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these KV channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that KV1.2, KV1.4, KV1.5, and KV2.2 are expressed at the protein level in GI tissues and smooth muscle cells. KV2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of KV channel subunits.
Nitrergic regulation of colonic transit in rats
Authors: Mizuta Yohei , Takahashi Toku , Owyang Chung
Published: December 2017

Nitric oxide has been shown to be an inhibitory neurotransmitter in the mammalian colon, although its role in colonic transit remains unclear. We investigated the effect of the nitric oxide biosynthesis inhibitor NG -nitro-l-arginine methyl ester (l-NAME) on colonic transit in conscious rats. Colonic transit was determined by calculating the geometric center of the distribution of radiochromium instilled into the proximal colon. We also studied the effect ofl-NAME on colonic motility in vivo and on descending relaxation in vitro.l-NAME (10 mg/kg) significantly delayed colonic transit compared with saline. The inhibitory effect ofl-NAME was prevented byl-arginine (100 mg/kg) but not by d-arginine (100 mg/kg).l-NAME (10 mg/kg) induced random and uncoordinated phasic contractions throughout the rat colon in vivo. Luminal distension evoked descending relaxation in the proximal and distal rat colon in vitro.l-NAME (10−4 M) significantly inhibited this relaxation. It is suggested, therefore, that nitric oxide enhances transit in the rat colon by mediating descending relaxation, which, in turn, facilitates propulsion of the colonic contents.
Substance P inhibits pancreatic exocrine secretion via a neural mechanism
Authors: Kirkwood Kimberly S. , Kim Edward H. , He Xiao Dong , Calaustro Edna Q. , Domush Christopher , Yoshimi Shandra K. , Grady Eileen F. , Maa John , Bunnett Nigel W. , Debas Haile T.
Published: December 2017

We investigated the effects of the sensory neuropeptide substance P (SP) on amylase and fluid secretion in the isolated vascularly perfused rat pancreas. SP inhibited CCK-induced amylase release and secretin-induced juice flow via the pancreatic duct in a dose-related fashion. Threshold inhibition occurred following addition of 10−10M SP to the perfusate, and maximal inhibition was seen with 10−8M SP. The effects of SP were partially blocked by both the neurokinin-1 (NK1) and neurokinin-2 (NK2) receptor antagonists. Atropine and TTX blocked SP-induced effects on both amylase secretion (26 and 63% blockade, respectively) and pancreatic juice flow (21 and 79% blockade, respectively). Excitation of pancreatic sensory nerves using capsaicin (in the absence of SP) inhibited both amylase and pancreatic juice flow via activation of the NK1 receptor. We conclude that SP inhibits exocrine secretion via an indirect neural mechanism.
Effects of calcium channel antagonists on LPS-induced hepatic iNOS expression
Authors: Mustafa Shamimunisa B. , Olson Merle S.
Published: December 2017

The onset of liver injury is a pivotal event during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer cells (KC), the resident macrophages of the liver, to generate an abundance of inflammatory substances, including nitric oxide (NO). Elevated levels of NO are thought to contribute to the propagation of liver injury during sepsis. Calcium, a major second messenger in several cellular signaling events, is required by the KC for the generation of inducible nitric oxide synthase (iNOS). The purpose of this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and to evaluate their effects on the regulation of iNOS expression in cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response was accompanied by an increase in iNOS mRNA formation in the intact liver. Pretreatment of rats with calcium channel antagonists (i.e., diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the serum ALT level and iNOS mRNA expression in the liver. Pretreatment of cultured KC with calcium channel antagonists for 1 h followed by the addition of LPS markedly repressed iNOS protein and mRNA expression. Time-course studies revealed that calcium channel antagonists were most effective at inhibiting LPS-induced iNOS mRNA formation by KC when added before LPS. Treatment of KC with calcium channel antagonists prior to the addition of LPS decreased nuclear levels of the p65 subunit of nuclear factor-κB and prevented the LPS-dependent degradation of the inhibitory protein IκBα. Thus our findings indicate that under endotoxemic conditions calcium channel antagonists limit hepatocellular injury that is accompanied by an inhibition of LPS-mediated iNOS expression in rat liver KC.
NSP4 elicits age-dependent diarrhea and Ca2+mediated I−influx into intestinal crypts of CF mice
Authors: Morris Andrew P. , Scott Jason K. , Ball Judith M. , Zeng Carl Q.-Y. , O’Neal Wanda K. , Estes Mary K.
Published: December 2017

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4114–135peptide, can overcome these gastrointestinal complications in CF (CFTRm3Baynull mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR+/+), heterozygous (CFTR+/−), or homozygous (CFTR−/−) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca2+mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I−influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR−/−background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca2+or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca2+-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4114–135to counteract chronic constipation/obstructive bowel syndrome in CF patients.
Expression and distribution of the Na+- HCO 3 − cotransporter in human pancreas
Authors: Marino Christopher R. , Jeanes Virginia , Boron Walter F. , Schmitt Bernhard M.
Published: December 2017

The cellular mechanisms of [Formula: see text] secretion in the human pancreas are unclear. Expression of a Na+-[Formula: see text]cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single ∼130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na+-K+pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of[Formula: see text] by human pancreatic duct cells involves the basolateral uptake of Na+and[Formula: see text] via NBC, an electrogenic Na+-[Formula: see text]cotransporter.
Role of inducible nitric oxide synthase in the regulation of VCAM-1 expression in gut inflammation
Authors: Kawachi Shigeyuki , Cockrell Adam , Laroux F. Stephen , Gray Laura , Granger D. Neil , van der Heyde Henri C. , Grisham Matthew B.
Published: December 2017

The objectives of this study were to assess the role of the inducible isoform of nitric oxide synthase (iNOS) on vascular cell adhesion molecule 1 (VCAM-1) expression in vivo in an acute model of inflammation induced in iNOS-deficient (iNOS−/−) mice and compare these data to those obtained by pharmacological inhibition of iNOS in a CD4+T lymphocyte-dependent model of chronic colitis. VCAM-1 expression was quantified in vivo using the dual radiolabel monoclonal antibody technique. We found that intraperitoneal injection of 10 μg/kg tumor necrosis factor-α (TNF-α) enhanced VCAM-1 expression by approximately twofold in the colon, cecum, and stomach but not small intestine in iNOS−/−mice compared with TNF-α-injected wild-type mice. Injection of wild-type mice with 25 μg/kg TNF-α further enhanced VCAM-1 expression by approximately twofold compared with wild-type mice injected with 10 μg/kg TNF-α; however, VCAM-1 expression was not further enhanced in any gastrointestinal organ system in iNOS−/−mice. In a second series of experiments, we found that continuous inhibition of iNOS using oral administration of NG-iminoethyl-l-lysine did not alter the enhanced levels of VCAM-1 expression in the colon nor did it alter the severity of colonic inflammation in SCID mice reconstituted with CD4+, CD45RBhighT cells. We conclude that iNOS may regulate VCAM-1 expression in acute inflammation; however, this effect is modest and tissue specific and occurs only when VCAM-1 expression is submaximal. iNOS does not appear to modulate VCAM-1 expression in an immune model of chronic colitis.
Cryptosporidium parvuminduces apoptosis in biliary epithelia by a Fas/Fas ligand-dependent mechanism
Authors: Chen Xian-Ming , Gores Gregory J. , Paya Carlos V. , LaRusso Nicholas F.
Published: December 2017

Although the clinical features of sclerosing cholangitis from opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which associated pathogens, such as Cryptosporidium parvum, cause disease are obscure. Using an in vitro model of biliary cryptosporidiosis, we observed that C. parvum induces apoptosis in cultured human biliary epithelia. Both caspase protease inhibitors and neutralizing antibodies to either Fas receptor (Fas) and Fas ligand (FasL) inhibited this process; neutralizing antibodies to other apoptotic cytokines [interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β)] had no effect. C. parvum stimulated FasL membrane surface translocation, increased both FasL and Fas protein expression in infected biliary epithelia, and induced a marked increase of soluble FasL (but not IL-1β, TNF-α, and TGF-β) in supernatants from infected cells. When a coculture model is used in which infected and uninfected cell populations were physically separated by a semipermeable membrane, both uninfected biliary epithelia and uninfected Fas-sensitive Jurkat cells (but not a Fas-resistant Jurkat cell line) underwent apoptosis when cocultured with infected biliary epithelia. Moreover, both a neutralizing antibody to FasL and a metalloprotease inhibitor blocked the apoptosis in uninfected cocultured cells. Activation of caspase activity was also observed in uninfected cocultured biliary epithelia. The data suggest that C. parvuminduces apoptosis in biliary epithelia by a Fas/FasL-dependent mechanism involving both autocrine and paracrine pathways. These observations may be relevant to both the pathogenesis and therapy of the cholangitis seen in AIDS patients with biliary cryptosporidiosis.
Carboxyl ester lipase activity in milk prevents fat-derived intestinal injury in neonatal mice
Authors: Howles Philip N. , Stemmerman Grant N. , Fenoglio-Preiser Cecilia M. , Hui David Y.
Published: December 2017

Carboxyl ester lipase (bile salt-stimulated lipase) is a pancreatic enzyme capable of hydrolyzing esters of cholesterol and fat-soluble vitamins. It also efficiently digests triglycerides (TG) into free fatty acids and glycerol and is abundant in the milk of humans and several other species. We used the mouse as a model to test the hypothesis that milk-derived carboxyl ester lipase (CEL) digests milk TG and that without its activity milk lipids and their digestion intermediates can disrupt the intestinal epithelium of neonates. CEL protein and enzymatic activity were shown to be abundant in mouse milk. After 24-h administration of the CEL-specific inhibitor, WAY-121,751–5, the small intestines of treated and control neonates were analyzed histologically for signs of fat malabsorption and injury to their villus epithelium. In vehicle-fed controls, TG were digested and absorbed in the duodenum and jejunum, whereas, in inhibitor-fed littermates, large intracellular neutral lipid droplets accumulated in enterocytes of the ileum, resulting in damage to the villus epithelium. Similar results were observed in neonates nursed by CEL knockout females compared with heterozygous controls. The results suggest that lack of CEL activity causes incomplete digestion of milk fat and lipid accumulation by enterocytes in the ileum of neonatal mice.
Marked increase of guanylin secretion in response to salt loading in the rat small intestine
Authors: Kita Toshihiro , Kitamura Kazuo , Sakata Junichiro , Eto Tanenao
Published: December 2017

Guanylin and uroguanylin are intestinal peptides that stimulate guanylate cyclase C and cause chloride secretion. These peptides show topological instability due to two disulfide bonds. The disulfide bonds were reduced and S-carboxymethylated to cleave the bonds and obtain stable and sole derivatives. We established a new and reliable RIA system for the stable derivatives from both peptides. With the use of this system, the response of the peptides to salt loading of the rat small intestine was evaluated. The lumen of the small intestines of Sprague-Dawley rats was perfused in vivo with Krebs-Ringer solution containing different concentrations of salt or mannitol. Mature guanylin, proguanylin, and mature uroguanylin were found in the perfusate in the basal state. The highest salt loading (200 mM NaCl for 20 min) increased the guanylin secretion about threefold (1.9 ± 0.2 vs. 5.4 ± 0.5 pmol/min), with the effect lasting for 60 min. The uroguanylin secretion was less affected. Hyperosmolar mannitol also caused a significant but smaller increase of guanylin secretion. Increased guanylin could lead to increased salt and water secretion of the intestine; thus members of the guanylin family have potential roles in the regulation of water and salt metabolism in the small intestine.
Relationship between terminal ileal pressure waves and propagating proximal colonic pressure waves
Authors: Dinning P. G. , Bampton P. A. , Kennedy M. L. , Cook I. J.
Published: December 2017

The relationship between propagating distal ileal and proximal colonic motor patterns has not been systematically examined in humans. Our aim was to define the relationships, if any, between prolonged propagating contractions or discrete clustered contractions and cecal propagating sequences using multiple manometric recording sites spanning the distal ileum and unprepared colon. In 14 healthy volunteers, a 17-lumen-perfused silicon catheter was positioned pernasally such that at least three recording sites lay in the ileum and the remainder in the colon. Intersidehole distance was 7.5 cm. In 271 h of recording, 30% of the terminal ileal propagating events was temporally associated with cecal propagating sequences. Significantly more prolonged propagating contractions (11 of 24, 46%; P = 0.02) were associated with cecal propagating sequences than were associated with ileal discrete clustered contractions (4 of 26, 15%). This trend was more pronounced at night. Of 159 cecal propagating sequences, 15 (9%) were preceded by an ileal propagating event. The remaining 91% was preceded by increased nonpropagating activity commencing 2 min before the cecal propagating sequence ( P = 0.0002). We conclude that distal ileal propagating motor patterns are one stimulus for cecal propagating sequences, with the association being stronger for prolonged propagating contractions than for discrete clustered contractions.
The cytoskeleton of digestive epithelia in health and disease
Authors: Ku Nam-On , Zhou Xiangjun , Toivola Diana M. , Omary M. Bishr
Published: December 2017

The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.
CEP-1347 inhibits caerulein-induced rat pancreatic JNK activation and ameliorates caerulein pancreatitis
Authors: Wagner Andreas C. C. , Mazzucchelli Luca , Miller Matthew , Camoratto Anna Marie , Göke Burkhard
Published: December 2017

Pancreatic caerulein-induced activation of c-Jun NH2-terminal kinase (JNK) has been reported, and JNK has been proposed as a mediator during induction of hyperstimulated pancreatitis. CEP-1347 has recently been described as a specific JNK inhibitor. We tested whether CEP-1347 inhibits caerulein-induced pancreatic JNK activation in isolated acini and in vivo. CEP-1347 dose dependently inhibited acinar caerulein-induced JNK activation with nearly complete inhibition at 2 μM but had no effect on digestive enzyme release. For in vivo studies, rats were pretreated with CEP-1347 before caerulein hyperstimulation. For assessment of JNK activation and histological alterations, animals were killed 30 min or 2 and 4 h after caerulein hyperstimulation, respectively. Pancreatic wet weight, serum enzyme levels, and pancreatic activity of p38 and extracellular signal-regulated kinase (ERK) were also determined. Caerulein hyperstimulation strongly activated JNK, p38, and ERK. CEP-1347 pretreatment dose dependently reduced caerulein-induced pancreatic JNK activation without p38 or ERK inhibition. JNK inhibition also reduced pancreatic edema formation and reduced histological severity of pancreatitis. Thus we show that CEP-1347 inhibits JNK activation in vivo and ameliorates caerulein-induced pancreatitis.